U.S. patent application number 17/265755 was filed with the patent office on 2021-08-19 for epitope of epb41l5, and monoclonal antibody.
The applicant listed for this patent is REPUREUS INC.. Invention is credited to Jae Ho CHEONG, Kyung Chul CHOI, Mi Hyeon JEONG, Ho Geun YOON.
Application Number | 20210252145 17/265755 |
Document ID | / |
Family ID | 1000005593292 |
Filed Date | 2021-08-19 |
United States Patent
Application |
20210252145 |
Kind Code |
A1 |
CHOI; Kyung Chul ; et
al. |
August 19, 2021 |
EPITOPE OF EPB41L5, AND MONOCLONAL ANTIBODY
Abstract
In the present disclosure, the effect of the
TGF.beta./Smad3/EPB41L5 molecular mechanism on cancer cells has
been identified, and it has been found that high expression of
EPB41L5 is correlated with poor overall survival of cancer
patients, indicating that EPB41L5 is a potential prognostic marker
of cancer. Thus, the present disclosure specifies an epitope of
EPB41L5 to allow EPB41L5 to be recognized as an antigen, and
relates to an antibody or a fragment thereof which binds
specifically to the epitope. The antibody according to the present
disclosure may be usefully employed as a therapeutic agent for
EPB41L5-related cancer.
Inventors: |
CHOI; Kyung Chul;
(Yangcheon-gu Seoul, KR) ; YOON; Ho Geun;
(Goyang-si Gyeonggi-do, KR) ; CHEONG; Jae Ho;
(Gangnam-gu Seoul, KR) ; JEONG; Mi Hyeon;
(Jeonju-si Jeollabuk-do, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
REPUREUS INC. |
Seodaemun-gu, Seoul |
|
KR |
|
|
Family ID: |
1000005593292 |
Appl. No.: |
17/265755 |
Filed: |
August 8, 2019 |
PCT Filed: |
August 8, 2019 |
PCT NO: |
PCT/KR2019/009960 |
371 Date: |
February 3, 2021 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 39/39558 20130101;
G01N 33/574 20130101; A61P 35/00 20180101; C07K 16/18 20130101;
C12N 15/113 20130101 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 35/00 20060101 A61P035/00; C07K 16/18 20060101
C07K016/18; C12N 15/113 20060101 C12N015/113 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 9, 2018 |
KR |
10-2018-0093045 |
Claims
1. A 1- to 6-mer epitope selected from among 619.sup.th to
624.sup.th amino acid residues of an EPB41L5 (Erythrocyte Membrane
Protein Band 4.1 Like 5) protein represented by the amino acid
sequence of SEQ ID NO: 1.
2. The epitope of claim 1, consisting of the 619.sup.th to
624.sup.th amino acid residues of the EPB41L5 protein.
3. A monoclonal antibody or a fragment thereof which recognizes an
EPB41L5 protein represented by the amino acid sequence of SEQ ID
NO: 1 as an antigen and binds specifically thereto.
4. The monoclonal antibody or fragment thereof of claim 3, which
binds to a 1- to 6-mer epitope selected from among 619.sup.th to
624.sup.th amino acid residues of the EPB41L5 protein represented
by the amino acid sequence of SEQ ID NO: 1.
5. The monoclonal antibody or fragment thereof of claim 3, which
comprises: a heavy-chain variable region comprising a heavy-chain
CDR1 represented by SEQ ID NO: 6, a heavy-chain CDR2 represented by
SEQ ID NO: 7, and a heavy-chain CDR3 represented by SEQ ID NO: 8;
and a light-chain variable region comprising a light-chain CDR1
represented by SEQ ID NO: 9, a light-chain CDR2 represented by SEQ
ID NO: 10, and a light-chain CDR3 represented by SEQ ID NO: 11.
6. The monoclonal antibody or fragment thereof of claim 3, which
blocks interaction between EPB41L5 and TGF-.beta.1.
7. The monoclonal antibody or fragment thereof of claim 3, wherein
the antibody is a chimeric antibody, a humanized antibody, a
bivalent-bispecific molecule, a minibody, a domain antibody, a
bispecific antibody, an antibody mimic, a diabody, a triabody, a
tetrabody, or a fragment thereof.
8. A nucleic acid molecule encoding the monoclonal antibody or
fragment thereof of claim 3.
9. A vector comprising the nucleic acid molecule of claim 8.
10. A host cell comprising the vector of claim 9.
11. A vaccine composition for preventing or treating cancer, the
vaccine composition containing, as an active ingredient: a 1- to
6-mer epitope selected from among 619.sup.th to 624.sup.th amino
acid residues of an EPB41L5 protein represented by the amino acid
sequence of SEQ ID NO: 1; a nucleic acid molecule encoding the
epitope; or a vector comprising the nucleic acid molecule.
12. The vaccine composition of claim 11, wherein the cancer is a
cancer stem cell.
13. A pharmaceutical composition for preventing or treating cancer,
the pharmaceutical composition containing, as an active ingredient,
at least any one selected from the group consisting of: the
monoclonal antibody or fragment thereof that recognizes an EPB41L5
protein represented by the amino acid sequence of SEQ ID NO: 1 as
an antigen and binds specifically thereto; a nucleic acid molecule
encoding the monoclonal antibody or fragment thereof; a vector
comprising the nucleic acid molecule; or an inhibitor that inhibits
expression of EPB41L5 gene.
14. The pharmaceutical composition of claim 13, wherein the
inhibitor that inhibits expression of EPB41L5 gene is at least one
selected from the group consisting of siRNA, shRNA, miRNA,
ribozyme, DNAzyme, peptide nucleic acid (PNA), and antisense
oligonucleotides.
15. The pharmaceutical composition of claim 14, wherein the siRNA
is an siRNA represented by the nucleotide sequence of SEQ ID NO: 4
or an siRNA represented by the nucleotide sequence of SEQ ID NO:
5.
16-23. (canceled)
24. A method for treating or preventing cancer, the method
comprising administering to a patient the pharmaceutical
composition according to claim 13.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a U.S. National Stage entry of
International Patent Application no. PCT/KR2019/09960, filed Aug.
8, 2019, which claims the benefit of priority of Korean Patent
Application no. 10-2018-0093045, filed Aug. 9, 2018.
TECHNICAL FIELD
[0002] The disclosure relates to an epitope of EPB41L5 (Erythrocyte
Membrane Protein Band 4.1 Like 5) protein and a monoclonal antibody
that binds specifically thereto.
BACKGROUND ART
[0003] Cancer is a very fatal disease that can threaten the life of
an individual by causing tissue cells to proliferate abnormally and
unlimitedly to form a tumor that prevents the organ from performing
its normal function. In 2017, the first leading cause of death in
Korean was malignant neoplasm (cancer), and 27.6% of the total
deaths were due to cancer.
[0004] Among cancer diseases, gastric cancer is one of the most
major causes of death. In particular, metastatic gastric cancer is
known as a malignant disease with a 5-year survival rate of less
than 30% in the world. Although studies related to new biomarkers
have been conducted for the treatment of such metastatic gastric
cancer, the specific pathogenesis of the metastatic gastric cancer
is not clearly known.
[0005] Targeted therapeutic agents developed to date include
HER2-targeting therapeutic agents, VEGFR2-targeting therapeutic
agents, and the like. In particular, Genetech/Roche found that HER2
is associated with a poor prognosis of breast cancer, ovarian
cancer or gastric cancer, and thus Genetech/Roche developed a
humanized monoclonal antibody (Herceptin or Trastuzumab) that binds
to the extracellular domain of HER2/neu. In addition,
Herceptin/Trastuzumab is a blockbuster drug that is currently sold
worldwide, and recorded sales of $950 million in the United States
only in the first half of 2011. However, Herceptin/Trastuzumab is
known to have disadvantages in that it does not exhibit a clinical
effect for other cancers that significantly overexpress HER2/neu,
except for breast cancer, and in that patients who have received
long-term administration of Herceptin/Trastuzumab have resistance
to Herceptin/Trastuzumab.
[0006] Therefore, there are emerging needs to identify biomolecules
related to cancer growth and metastasis and to discover biomarkers
for new cancer-targeting therapies controlling cancer growth and
metastasis by targeting these biomolecules.
[0007] The information disclosed in the above "Background Art"
section are only for enhancement of understanding of the background
of the present disclosure and should not be taken as an
acknowledgement that this information forms a conventional art
already known to a person skilled in the art.
DISCLOSURE
Technical Problem
[0008] An object of the present disclosure is to provide a 1- to
6-mer epitope selected from among the 619.sup.th to 624.sup.th
amino acid residues of an EPB41L5 protein represented by the amino
acid sequence of SEQ ID NO: 1.
[0009] Another object of the present disclosure is to provide a
monoclonal antibody or a fragment thereof that recognizes the
EPB41L5 protein represented by the amino acid sequence of SEQ ID
NO: 1 as an antigen and binds specifically thereto.
[0010] Still another object of the present disclosure is to
provide: a nucleic acid molecule encoding the monoclonal antibody
or fragment thereof; and a vector comprising the nucleic acid
molecule.
[0011] Yet another object of the present disclosure is to provide a
vaccine composition for preventing or treating cancer, the
composition containing, as an active ingredient: a 1- to 6-mer
epitope selected from among the 619.sup.th to 624.sup.th amino acid
residues of an
[0012] EPB41L5 protein represented by the amino acid sequence of
SEQ ID NO: 1; a nucleic acid molecule encoding the epitope; or a
vector comprising the nucleic acid molecule.
[0013] A further object of the present disclosure is to provide a
pharmaceutical composition for preventing, alleviating or treating
cancer, the composition containing, as an active ingredient: the
above-described monoclonal antibody or fragment thereof; a nucleic
acid molecule encoding the same; or a vector comprising the nucleic
acid molecule.
[0014] Further another object of the present disclosure is to
provide a method of providing information for diagnosis of a
disease caused by overexpression of EPB41L5.
[0015] Other objects and advantages of the present disclosure will
be more clearly understood from the following detailed description
of the present disclosure, the appended claims and the accompanying
drawings.
Technical Solution
[0016] The present inventors have found that overexpression of
EPB41L5 (Erythrocyte Membrane Protein Band 4.1 Like 5) protein
compared to that in a normal control group is associated with
cancer growth and metastasis, and have made extensive efforts to
discover a biomarker for new cancer-targeting therapy capable of
controlling cancer growth and metastasis by targeting the EPB41L5
protein. As a result, the present inventors have identified an
epitope of EPB41L5 protein that inhibits cancer growth and
metastasis through TGF.beta.-EPB41L5 signaling, thereby completing
the present disclosure.
[0017] According to one aspect of the present disclosure, the
present disclosure provides a 1- to 6-mer epitope selected from
among the 619.sup.th to 624.sup.th amino acid residues of an
EPB41L5 protein represented by the amino acid sequence of SEQ ID
NO: 1.
[0018] In the present disclosure, the EPB41L5 protein may be
represented by SEQ ID NO: 1, and information on the sequence
thereof may be found in GenBank No. NM_020909.
[0019] In the present disclosure, the EPB41L5 (Erythrocyte membrane
protein band 4.1 like 5) protein belongs to the NBL4 subgroup of
the Erythrocyte Membrane Protein Band 4.1 superfamily, which has an
FERM domain at the N-terminus and a non-homologous sequence at the
C-terminus.
[0020] In the present specification, the term "epitope" refers to a
localized region of an antigen to which an antibody or a fragment
thereof may specifically bind. Epitopes usually consist of surface
groups of molecules such as amino acids or sugar side chains, and
usually have specific three-dimensional structural characteristics,
as well as specific charge characteristics. Conformational and
non-conformational epitopes are distinguished in that the binding
to the conformational epitope but not the non-conformational
epitope is lost in the presence of denaturing solvents. The epitope
may comprise amino acid residues directly involved in the binding
(also called immunodominant component of the epitope) and other
amino acid residues, which are not directly involved in the
binding, such as amino acid residues which are effectively blocked
by the specific antigen binding peptide (that is, the amino acid
residue is within the footprint of the specific antigen binding
peptide).
[0021] In the present disclosure, the epitope may comprise any one
or more amino acids selected from among the amino acid residues at
positions 619, 620, 621, 622, 623 and 624 of the EPB41L5
protein.
[0022] In the present disclosure, the epitope may consist of the
619.sup.th to 624.sup.th amino acids of the EPB41L5 protein, and
may preferably consist of an amino acid sequence represented by SEQ
ID NO: 2, but is not limited thereto.
[0023] In the present disclosure, the epitope containing the amino
acids at the above-described positions, when used as a vaccine or
composition, may be used in combination with a carrier so that the
structure thereof is maintained. Although the carrier according to
the present disclosure is not particularly limited, as long as it
is biocompatible and may achieve the desired effect in the present
disclosure, it may preferably be selected from among peptides,
serum albumin, immunoglobulins, hemocyanin, and
polysaccharides.
[0024] According to another aspect of the present disclosure, the
present disclosure provides a monoclonal antibody or a fragment
thereof that recognizes the EPB41L5 protein represented by SEQ ID
NO: 1 as an antigen and binds specifically thereto.
[0025] According to a preferred embodiment of the present
disclosure, the antibody or fragment thereof according to the
present disclosure may bind to a 1- to 6-mer epitope selected from
among the 619.sup.th to 624.sup.th amino acids of EPB41L5
protein.
[0026] Epithelial-mesenchymal transition (EMT) is a primary cause
of gastric cancer metastasis, and many studies have reported that
the TGF.beta. signaling regulates epithelial-mesenchymal
transition. TGF.beta. ligand binds to TGF.beta. receptor I through
the serine/threonine kinase TGF.beta. receptor II. The TGF.beta.
receptor dimer is activated by the phosphorylation of Smad2/3 and,
in turn, the phosphorylated Smad2/3 binds to the co-Smad Smad4, and
this protein complex enters the nucleus and regulates
epithelial-mesenchymal transition-related genes such as PAI-1,
ZEB1, Slug, and like. Some studies reported that TGF-.beta.1
expression is enhanced in the mucosa, serum and tissue of gastric
cancer patients, and that high expression of TGF-.beta.1 is
involved in the low survival rate of gastric cancer patients.
[0027] In the present disclosure, it was confirmed through
oligonucleotide microarray analysis and Kaplan-Meier survival
analysis that the survival rate of cancer patients was low when the
expression levels of EPB41L5 gene and a protein encoded thereby
were high. In addition, it was confirmed that EPB41L5 mRNA and
protein expressions in four gastric cancer cell lines (KATOIII,
MKN28, SNU1, and SNU719) were increased by
[0028] TGF-.beta.1, and that the increased EPB41L5 expression
induced by TGF-.beta.1 was regulated by Smad-dependent TGF.beta.
signaling. The increased EPB41L5 protein expression induced by
TGF.beta. signaling may affect cancer metastasis by interaction
with the cell adhesion molecule p120-catenin. Accordingly, the
monoclonal antibody or fragment thereof according to the present
disclosure binds specifically to an epitope that is an amino acid
sequence which is bound when an EPB41L5/p120-catenin complex is
formed, and thus the monoclonal antibody or fragment thereof may
very effectively inhibit cancer occurrence, metastasis or growth by
effectively inhibiting the formation of the EPB41L5/p120-catenin
complex.
[0029] According to a preferred embodiment of the present
disclosure, the monoclonal antibody and fragment thereof binds
specifically to EPB41L5 represented by SEQ ID NO: 1 and blocks the
interaction between EPB41L5 and p120-catenin. Although the
monoclonal antibody and fragment thereof is not particularly
limited to as long as it is produced using the EPB41L5 protein
represented by SEQ ID NO: 1 as an antigen, it is most preferably
produced using the amino acid sequence of SEQ ID NO: 2 as an
antigen, but is not limited thereto.
[0030] In the present specification, the term "epitope" refers to a
localized region of an antigen to which an antibody or a fragment
thereof may specifically bind. An epitope may be, for example,
contiguous amino acids of a polypeptide as an antigen, or an
epitope may come together from two or more non-contiguous regions
juxtaposed by tertiary folding in a polypeptide. An epitope may
comprise at least 2, at least 3, at least 4, at least 5, at least
6, at least 7, at least 8, at least 9, at least 10, at least 11, at
least 12, at least 13, at least 14 or at least 15 contiguous or
non-contiguous amino acids in the unique three-dimensional
structure of the antigen. The antibody or fragment thereof
according to the present disclosure recognizes EPB41L5 as an
antigen and binds specifically thereto. Specifically, the antibody
or fragment thereof may specifically bind to a 1- to 6-mer epitope
selected from among the 619.sup.th to 624.sup.th amino acid
residues of EPB41L5, and the epitope may comprise one or more amino
acids.
[0031] Methods for determining an epitope to which a given antibody
binds (e.g., epitope mapping) include various methods such as
immunoblotting and immunoprecipitation assays based on antibody
reactivity tests. The three-dimensional spatial structure of the
epitope may be determined using various methods such as x-ray
crystallography, 2-dimensional nuclear magnetic resonance and
HDX-MS (Epitope Mapping Protocols in Methods in Molecular Biology,
Vol. 66, G. E. Morris, Ed. (1996)).
[0032] According to a preferred embodiment of the present
disclosure, an epitope to which the antibody or fragment thereof
according to the present disclosure may bind may be determined by
NMR spectroscopy, X-ray diffraction crystallography, ELISA assay,
HDX-MS (hydrogen/deuterium exchange coupled with mass
spectrometry), array-based oligo-peptide scanning assays, and/or
mutagenesis mapping (Giege R et al., (1994) Acta Crystallogr D Biol
Crystallogr 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem
189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A
(1976) J Biol Chem 251: 6300-6303).
[0033] As used herein, the term "antibody" may be any type of
antibody (e.g., IgG, IgE, IgM, IgD, IgA, or IgY) among
immunoglobulin molecules, or may be any isotype of antibody (e.g.,
IgG1, IgG2, IgG3, and IgG4 in humans; and IgG1, IgG2a, IgG2b, and
IgG3 in mice). Immunoglobulins (e.g., IgG1) may exist in several
allotypes, and the term "antibody" as used herein includes
generally known isotypes and allotypes. In addition, the term
"antibody" as used herein may be IgG1, IgG2, IgG3, IgG4, or a
hybrid thereof (e.g., a hybrid of IgG2 and IgG4).
[0034] As used herein, the term "monoclonal antibody" refers to an
antibody that displays single binding specificity and affinity for
a particular epitope.
[0035] The monoclonal antibody of the present disclosure may be
produced, for example, by the hybridoma method first described in
Kohler et al., Nature 256, 495 (1975), or by a recombinant DNA
method. In addition, the monoclonal antibody may be isolated from a
phage antibody library using, for example, techniques described in
Clackson et al., Nature 352, 624-628 (1991) and Marks et al., J.
Mol. Biol. 222, 581-597 (1991). Monoclonal antibodies may be
obtained from any suitable source. The monoclonal antibody in the
present disclosure may be obtained from hybridomas produced either
from cells expressing EPB41L5 antigen or from cells obtained from
mice immunized with the antigen of interest in the form of a
nucleic acid encoding EPB41L5 antigen. The monoclonal antibody may
also be obtained from hybridomas derived from antibody-expressing
cells of immunized humans or non-human mammals such as rats, dogs,
primates, and the like.
[0036] In the present specification, the monoclonal antibody is
used in the sense of including a fragment thereof, and the fragment
preferably refers to an antigen-binding fragment. The fragment may
be produced using various methods known in the art. For example,
Fab and F(ab')2 fragments may be produced by proteolytic cleavage
of immunoglobulin molecules using enzymes such as papain
(production of Fab fragment) or pepsin (production of (F(ab')2)
fragment).
[0037] As used herein, the term "fragment" may be Fab, Fab',
F(ab')2, Fv, scFV (single chain Fv), or sdAb containing a monomeric
VH or VL domain, and the fragment is well known in the art.
[0038] In the present disclosure, the antibody may be, but is not
limited to, a chimeric antibody, a humanized antibody, a
bivalent-bispecific molecule, a minibody, a domain antibody, a
bispecific antibody, an antibody mimic, a diabody, a triabody, a
tetrabody, or a fragment thereof.
[0039] In the present disclosure, the "chimeric antibody" is an
antibody obtained by recombining the variable region of a mouse
antibody and the constant region of a human antibody, and has a
significantly improved immunity compared to the mouse antibody.
[0040] In addition, in the present disclosure, the "humanized
antibody" refers to an antibody obtained by modifying the protein
sequence of an antibody, derived from a non-human species, so as to
be similar to that of an antibody variant naturally produced in
humans. For example, the humanized antibody may be produced by
recombining a mouse-derived CDR with a human antibody-derived FR to
produce a humanized variable region and recombining the same with a
desired human antibody constant region.
[0041] The monoclonal antibody or fragment thereof according to the
present disclosure inhibits physiological effects associated with
the growth and metastasis of cancer mediated by the EPB41L5 gene or
the protein encoded thereby. Specifically, increased TGF-.beta.1
expression in cancer is identified and the expression level of the
EPB41L5 protein is increased by TGF-.beta.1, but the expression
level of the EPB41L5 protein is regulated by Smad-dependent
TGF.beta. signaling. That is, increased EPB41L5 protein expression
level induced by TGF-.beta.1 affects the migration and invasion of
cancer through epithelial-mesenchymal transition, and cell
signaling mediated by the EPB41L5 protein may be very effectively
inhibited by treatment with the monoclonal antibody or fragment
thereof including the above-described antigen-binding protein.
[0042] As used herein, the expression "inhibit growth" is intended
to include any measurable decrease in cell growth when contacted
with the monoclonal antibody or fragment thereof, as compared to
the growth of the same cells not in contact with the monoclonal
antibody or fragment thereof of the present disclosure, for
example, an inhibition of growth of a cell culture by about 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. The decrease in
cell growth may be caused by various mechanisms.
[0043] The monoclonal antibody or fragment thereof contains one or
more CDRs (e.g., 1, 2, 3, 4, 5 or 6 CDRs). The monoclonal antibody
or fragment thereof, which binds to the EPB41L5 protein as an
antigen, is a polypeptide containing one or more
complementarity-determining regions (CDRs) as described herein. In
the antigen binding protein, the CDRs are oriented such that the
proper antigen binding properties of the CDR(s) are achieved. In
general, the antigen binding protein that is provided herein may
interfere with, block, reduce or modulate the interaction between
EPB41L5 and p120-catenin. That is, the antigen binding protein may
inhibit TGF-.beta.1-mediated cancer metastasis and growth by
inhibiting the formation of the EPB41L5/p120-catenin complex in a
subject.
[0044] In the present disclosure, the monoclonal antibody or
fragment thereof may comprise, but is not limited to:
[0045] a heavy-chain variable region comprising a heavy-chain CDR1
represented by SEQ ID NO: 6, a heavy-chain CDR2 represented by SEQ
ID NO: 7, and a heavy-chain CDR3 represented by SEQ ID NO: 8; and a
light-chain variable region comprising a light-chain CDR1
represented by SEQ ID NO: 9, a light-chain CDR2 represented by SEQ
ID NO: 10, and a light-chain CDR3 represented by SEQ ID NO: 11.
[0046] In the present disclosure, the monoclonal antibody or
fragment thereof may comprise, but is not limited to:
[0047] a heavy-chain variable region represented by SEQ ID NO: 12;
and a light-chain variable region represented by SEQ ID NO: 13.
[0048] A VH domain or one or more CDRs thereof may be linked to a
constant domain for forming a heavy chain. Similarly, a VL domain
or one or more CDRs thereof may be linked to a constant domain for
forming a light chain. A full-length heavy chain and a full-length
light chain combine to form a full-length antibody.
[0049] In the present disclosure, as described above, the
monoclonal antibody or fragment thereof, which binds to a 1- to
6-mer epitope selected from among the 619.sup.th to 624.sup.th
amino acid residues of the EPB41L5 protein represented by SEQ ID
NO: 1, binds specifically to the EPB41L5 represented by SEQ ID NO:
1, thereby exhibiting an effect of blocking the interaction between
EPB41L5 and p120-catenin.
[0050] In the present disclosure, the monoclonal antibody or
fragment thereof may be used for therapeutic applications.
Specifically, it may inhibit, suppress or modulate one or more
biological activities of the EPB41L5 gene or the protein encoded
thereby, and may bind specifically to the EPB41L5 protein, thereby
substantially inhibiting EPB41L5 protein-induced cell signaling
through competitive binding with a protein (e.g., TGF-.beta.1) that
may bind to the EPB41L5 protein. Thus, the monoclonal antibody or
fragment thereof may be very effectively used for therapeutic
applications.
[0051] Variable regions of immunoglobulin chains generally exhibit
the same overall structure comprising relatively conserved
framework regions (FR) joined by three hypervariable regions (more
often called "complementarity determining regions" or CDRs). The
CDRs from the two chains of each heavy chain/light chain pair
mentioned above are typically aligned by the framework regions to
form a structure that binds specifically to a specific epitope on
the target protein (e.g., PCSK9). From N-terminal to C-terminal,
naturally-occurring light and heavy chain variable regions both
typically conform with the following order of these elements: FR1,
CDR1, FR2, CDR2, FR3, CDR3 and FR4. A numbering system has been
devised for assigning numbers to amino acids that occupy positions
in each of these domains. This numbering system is defined in Kabat
Sequences of Proteins of Immunological Interest (1987 and 1991,
NIH, Bethesda, Md.)], or [Chothia & Lesk, 1987, J. Mol.
Bio1.196:901-917]; [Chothia et al., 1989, Nature 342:878-883.
[0052] Various heavy chain and light chain variable regions may be
provided herein, and as described above, each of these variable
regions may be attached to the above heavy and light chain constant
regions to form a complete antibody heavy and light chain,
respectively. Furthermore, each of the formed heavy and light chain
sequences may be combined to form a complete antibody
structure.
[0053] The monoclonal antibody or fragment thereof according to the
present disclosure may include a variant of the amino acid
sequence, as long as it may bind specifically to the EPB41L5. For
example, the amino acid sequence of the antibody may be modified to
improve the binding affinity and/or other biological properties of
the antibody. Such modifications include, for example, deletion,
insertion and/or substitution of one or more residues in the amino
acid sequence of the antibody.
[0054] Such amino acid variations are based on the relative
similarity of the amino acid side-chain substituents, for example,
their hydrophobicity, hydrophilicity, charge, size, and the like.
An analysis of the size, shape and type of the amino acid
side-chain substituents reveals that arginine, lysine, and
histidine are all positively charged residues; that alanine,
glycine and serine are all of a similar size; and that
phenylalanine, tryptophan and tyrosine all have a similar shape.
Therefore, based upon these considerations, arginine, lysine and
histidine; alanine, glycine and serine; and phenylalanine,
tryptophan and tyrosine; may be defined as biologically functional
equivalents.
[0055] To introduce variations, the hydropathic index of amino
acids may be considered. Each amino acid has been assigned a
hydropathic index on the basis of their hydrophobicity and charge
characteristics, which are as follows: isoleucine (+4.5); valine
(+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine
(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4);
threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine
(-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5);
glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine
(-3.9); and arginine (-4.5). The importance of the hydropathic
amino acid index in conferring interactive biological function on a
protein is generally understood in the art. It is known that
certain amino acids may be substituted for another amino acids
having a similar hydropathic index and still retain a similar
biological activity. To introduce variations based on the
hydropathic index, substitutions are made between amino acids that
exhibit a hydropathic index difference of preferably within .+-.2,
more preferably .+-.1, even more preferably .+-.0.5.
[0056] Meanwhile, it is also known that substitution between amino
acids having similar hydrophilicity values results in proteins
having equivalent biological activity. As disclosed in U.S. Pat.
No. 4,554,101, the following hydrophilicity values have been
assigned to amino acid residues: arginine (+3.0); lysine (+3.0);
aspartate (+3.0.+-.1); glutamate (+3.0.+-.1); serine (+0.3);
asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4);
proline (-0.5.+-.1); alanine (-0.5); histidine (-0.5); cysteine
(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8);
isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5);
tryptophan (-3.4). To introduce variations based on the hydropathic
value, substitutions are made between amino acids that exhibit a
hydropathic value difference of preferably within .+-.2, more
preferably .+-.1, even more preferably .+-.0.5.
[0057] Amino acid exchanges in proteins, which do not generally
alter the activity of molecules, are known in the art (H. Neurath,
R. L. Hill, The Proteins, Academic Press, New York (1979)). The
most commonly occurring exchanges are exchanges between amino acid
residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,
Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn,
Leu/Ile, Leu/Val, and Gln/Glu.
[0058] Considering the above-described variations having
biologically equivalent activity, it is interpreted that the
binding molecules of the present disclosure also include a sequence
showing substantial identity with the sequence set forth in the
Sequence Listing.
[0059] As used herein, the term "substantial identity" means a
sequence showing at least 61% homology, more preferably 70%
homology, even more preferably 80% homology, most preferably 90%
homology, as determined by aligning the sequence of the present
disclosure with any other sequence to correspond to each other as
much as possible and analyzing the aligned sequence using an
algorithm commonly used in the art. Alignment methods for sequence
comparison are known in the art. Various methods and algorithms for
alignment are disclosed in Smith and Waterman, Adv. Appl. Math.
2:482(1981); Needleman and Wunsch, J. Mol. Bio.48:443(1970);
Pearson and Lipman, Methods in Mol. Biol. 24: 307-31(1988); Higgins
and Sharp, Gene 73:237-44(1988); Higgins and Sharp, CABIOS
5:151-3(1989); Corpet et al., Nuc. Acids Res. 16:10881-90(1988);
Huang et al., Comp. Appl. BioSci. 8:155-65(1992) and Pearson et
al., Meth. Mol. Biol. 24:307-31(1994). NCBI Basic Local Alignment
Search Tool (BLAST) (Altschul et al., J. Mol. Biol.
215:403-10(1990)) is available from NCBI (National Center for
Biological Information), and may be used in conjunction with
sequencing programs, such as blastp, blasm, blastx, tblastn and
tblastx, on the Internet. BLAST is available from
http://www.ncbi.nlm.nih.gov/BLAST/. Sequence homology comparison
methods using this program can be identified online
(http://www.ncbi.nlm.nih.gov/BLAST/blast_help.html).
[0060] In the present disclosure, although the binding molecule,
preferably the antibody, may be produced by a conventional method
for producing an antibody, it may be produced by affinity
maturation.
[0061] As used herein, the term "affinity maturation" refers to a
process in which antibodies having increased affinity for an
antigen are produced by activated B cells in the course of an
immune response. For the purpose of the present disclosure, the
affinity maturation allows antibodies or antibody fragments to be
produced due to affinity maturation based on the principles of
mutation and selection, in the same process that occurs in
nature.
[0062] According to still another aspect of the present disclosure,
the present disclosure provides: a nucleic acid molecule encoding
the monoclonal antibody or fragment thereof; a vector comprising
the nucleic acid molecule; and a host cell comprising the
vector.
[0063] The nucleic acid molecule of the present disclosure may be
an isolated or recombinant nucleic acid molecule. Examples of such
nucleic acid molecules include single- and double-stranded DNA and
RNA and their corresponding complementary sequences. The "isolated
nucleic acid" may be isolated from a naturally occurring source. In
this case, the isolated nucleic acid is separated from the
peripheral gene sequence present in the genome of a subject from
which the nucleic acid was isolated. The isolated nucleic acid may
be understood as a nucleic acid, for example, a PCR product, a cDNA
molecule or an oligonucleotide, which is enzymatically or
chemically synthesized from a template. In this case, the nucleic
acid produced from this procedure can be understood as the isolated
nucleic acid molecule. The isolated nucleic acid molecule
represents a nucleic acid molecule in the form of a separate
fragment or as a component of a larger nucleic acid construct. A
nucleic acid is "operably linked" when arranged in a functional
relationship with another nucleic acid sequence. For example, the
DNA of a presequence or secretory leader is operably linked to the
DNA of the polypeptide when expressed as a preprotein, which is a
presecretory polypeptide. A promoter or an enhancer affecting the
transcription of the polypeptide sequence is operably linked to a
coding sequence, or a ribosome-binding site is operably linked to a
coding sequence when it is arranged such that translation is
promoted. Generally, the term "operably linked" means that DNA
sequences to be linked are located adjacent to each other. In the
case of secretory leaders, the term "operably linked" means that
the secretory leaders are present adjacent to each other in the
same leading frame. However, an enhancers does not located
adjacent. The linkage is performed by ligation at a convenient
restriction enzyme site. In the case where this site does not
exist, a synthetic oligonucleotide adaptor or a linker is used
according to a conventional method.
[0064] As used herein, the term "vector" refers to a carrier into
which a nucleic acid sequence can be inserted for introduction into
a cell where it can be replicated. The nucleic acid sequence can be
exogenous or heterologous.
[0065] As used herein, the term "expression vector" refers to a
vector containing a nucleic acid sequence encoding at least a
portion of a gene product capable of being transcribed. In some
cases, RNA molecules are then translated into a protein,
polypeptide, or peptide. Expression vectors may contain a variety
of control sequences. In addition to control sequences that
regulate transcription and translation, vectors and expression
vectors may contain nucleic acid sequences that provide other
functions as well.
[0066] In a specific embodiment of the present disclosure, the
expression vector may be selected from the group consisting of a
commercially widely available pCDNA vector, F, R1, RP1, Col,
pBR322, ToL, Ti vector; cosmids; phages such as lambda, lambdoid,
M13, Mu, p1, P22, Q.mu., T-even, T2, T3, T7, etc.; and plant
viruses, but is not limited thereto. Any expression vector known to
those skilled in the art may be used in the present disclosure, and
the choice of the expression vector is dependent on the nature of
the host cell of choice. Introduction of the vector into host cells
can be effected by, but not limited to, calcium phosphate
transfection, virus infection, DEAE-dextran mediated transfection,
lipofectamin transfection or electroporation, and any person
skilled in the art can select and use an introduction method
suitable for the expression vector and host cell used. Preferably,
the vector contains one or more selectable markers, but is not
limited thereto, and a vector containing no selectable marker may
also be used to determine whether a product has been produced. The
choice of the selectable marker may depend on the host cells of
choice, and the present disclosure is not limited thereto because
this choice is performed using a method already known to those
skilled in the art.
[0067] To facilitate the purification of the nucleic acid molecule
of the present disclosure, a tag sequence may be inserted and fused
into the expression vector. Examples of the tag include, but are
not limited to, a hexa-histidine tag, a hemagglutinin tag, a myc
tag or a flag tag. Any tag facilitating purification, known to
those skilled in the art, may be used in the present
disclosure.
[0068] As used herein, the term "host cell" includes eukaryotes and
prokaryotes, and refers to any transformable organism that is
capable of replicating the vector or expressing the gene encoded by
the vector. The host cell may be transfected or transformed by the
vector. The transfection or transformation refers to a process in
which the exogenous nucleic acid molecule is transferred or
introduced into the host cell.
[0069] Preferred examples of the host cell of the present
disclosure include, but are not limited to, bacterial cells, CHO
cells, HeLa cells, HEK293 cells, BHK-21 cells, COS7 cells, COP5
cells, A549 cells, NIH3T3 cells, etc.
[0070] According to yet another aspect of the present disclosure,
the present disclosure provides a vaccine composition for
preventing or treating cancer, the vaccine composition containing,
as an active ingredient: a 1- to 6-mer epitope selected from among
the 619.sup.th to 624.sup.th amino acid residues of an EPB41L5
protein represented by the amino acid sequence of SEQ ID NO: 1; a
nucleic acid molecule encoding the same; or a vector comprising the
same.
[0071] In the vaccine composition of the present disclosure, the
contents related to the EPB41L5, epitope, nucleic acid molecule and
vector are the same as described above, and thus description
thereof is omitted to avoid excessive complexity of the present
specification.
[0072] The vaccine may be a live vaccine, an attenuated vaccine or
an inactivated vaccine, and the vaccine may be used as a feed or
feed additive.
[0073] The vaccine composition of the present disclosure can
prevent or treat cancer by inducing an immune response as well as a
systemic immune response to EPB41L5 through active immunity. Active
immunity means that, when a pathogen invades, a living body is
immunized by generating antibodies in its own body.
[0074] In addition, the vaccine composition of the present
disclosure may be used to prevent or treat cancer by administering
the same to a subject.
[0075] The cancer in the present disclosure may be a cancer in
which the EPB41L5 gene or the protein encoded thereby is determined
to be overexpressed.
[0076] As used herein, the term "overexpression" means that the
expression level of the EPB41L5 gene or the protein encoded thereby
is 1.1 to 2 times higher than that in control cells (e.g., normal
cells of the organ of interest) when the expression level of
EPB41L5 is measured by an appropriate expression assay. The kind of
the cancer in the present disclosure is not limited, and the
vaccine composition of the present disclosure may be administered
to treat a number of cancers, including lymphomas such as leukemia,
acute lymphocytic leukemia, acute nonlymphocytic leukemia, chronic
lymphocytic leukemia, chronic myelogenous leukemia, Hodgkin's
disease, non-Hodgkin's lymphomas and multiple myeloma; childhood
solid tumors such as brain tumors, glioblastoma, neuroblastoma,
Rhabdomyosarcoma, retinoblastoma, Wilms tumor, bone tumors and
soft-tissue sarcomas; and common adult solid tumors such as lung
cancer, breast cancer, prostate cancer, urinary cancers, uterine
cancers, oral cancers, pancreatic cancer, melanoma, skin cancers,
stomach cancer, ovarian cancer, brain tumors, liver cancer,
laryngeal cancer, thyroid cancer, esophageal cancer and testicular
cancer.
[0077] The cancer in the present disclosure may be a cancer stem
cell.
[0078] As used herein, the term "cancer stem cell" refers, in a
broad sense, to cancer cells having self-renewal or differentiation
ability, which is the unique ability of stem cells. The cancer stem
cells are known to be present in tumors, and are believed to occur
due to abnormal metastasis of the genetic information of normal
stem cells. It is known that cancer stem cells are maintained and
proliferated due to the presence of microenvironments (niches) for
their survival, and that normal cells, immune-related cells or
differentiated cancer cells, which are present around cancer stem
cells, affect the maintenance of characteristics and proliferation
of these cancer stem cells. In the normal tumor growth conditions
of cancer stem cells (the "normal tumor growth conditions" refers
to a state in which a nutrient (glucose) required for cell growth
is sufficient and growth conditions for tumor microenvironments are
abundant, and thus there is no cell stress), the cancer stem cells
may proliferate at a slow rate, unlike common cancer cells, or may
be maintained in a dormant state, and thus may have resistance to
anticancer agents. For example, expression of transcription
regulators may be controlled, unlike that in normal tumor cells,
and thus the function of major metabolism regulatory substances
therein may differ from that in common cancer cells. Thus, the term
"cancer stem cells" generally refers to cells that acquire
resistance to apoptosis in a nutrient-deficient state through this
different metabolism regulatory ability and the regulation of cell
signaling systems mechanistically linked thereto, and have invasive
and/or metastatic potential. However, the cancer stem cells are not
limited thereto and may include any cells that may differentiate
into common cancer cells.
[0079] According to a further aspect of the present disclosure, the
present disclosure provides a composition containing: the
above-described monoclonal antibody or fragment thereof; a nucleic
acid molecule encoding the same; a vector comprising the nucleic
acid molecule; or an inhibitor that inhibits expression of the
EPB41L5 gene.
[0080] According to a preferred embodiment of the present
disclosure, the composition of the present disclosure may be used
as a pharmaceutical composition for prevention or treatment of
cancer or for inhibition of cancer metastasis.
[0081] In the composition of the present disclosure, contents
related to the antibody or fragment thereof, nucleic acid molecule,
cancer, cancer stem cell, etc. are the same as described above with
respect to in the epitope, monoclonal antibody or fragment thereof,
etc., and thus description thereof is omitted to avoid excessive
complexity of the present specification.
[0082] The pharmaceutical composition of the present disclosure may
comprise: (a) the antibody or fragment thereof; a nucleic acid
molecule encoding the same; a vector comprising the nucleic acid
molecule; or an inhibitor that inhibits expression of the EPB41L5
gene; and (b) a pharmaceutically acceptable carrier.
[0083] The greatest feature of the pharmaceutical composition of
the present disclosure is that the pharmaceutical composition
treats cancer or inhibits cancer metastasis by targeting the
EPB41L5 gene or the protein encoded thereby and inhibiting the
expression level of the EPB41L5 gene or the expression or activity
of the EPB41L5 protein.
[0084] The inhibitor of the present disclosure may be one or more
selected from the group consisting of siRNA (small interference
RNA), shRNA (short hairpin RNA), miRNA (microRNA), ribozyme,
DNAzyme, PNA (peptide nucleic acids), and antisense
oligonucleotides. Preferably, the inhibitor is siRNA (small
interference RNA) that binds specifically to the mRNA of the gene.
As the inhibitor that inhibits expression of the EPB41L5 gene, the
siRNA may be represented by the nucleotide sequence of SEQ ID NO: 4
or nucleotide sequence of SEQ ID NO: 5.
[0085] According to further another aspect of the present
disclosure, the present disclosure provides a method for preventing
or treating cancer, the method comprising a step of administering
the pharmaceutical composition.
[0086] In the method for preventing or treating cancer according to
the present disclosure, contents related to the pharmaceutical
composition are the same as described above with respect to the
pharmaceutical composition, and thus description thereof is omitted
to avoid excessive complexity of the specification.
[0087] As used herein, the term "prevention" may include, without
limitation, any action enabling the symptoms caused by cancer to be
blocked or inhibited or delayed using the composition of the
present disclosure.
[0088] As used herein, the term "treatment" may include, without
limitation, any action enabling the symptoms caused by cancer to be
alleviated or beneficially changed using the composition of the
present disclosure. The pharmaceutically acceptable carrier that is
contained in the vaccine composition and pharmaceutical composition
of the present disclosure is a carrier that is commonly used for
formulation, and examples thereof include, but are not limited to,
lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia,
calcium phosphate, alginate, gelatin, calcium silicate,
microcrystalline cellulose, polyvinyl pyrrolidone, cellulose,
water, syrup, methyl cellulose, methylhydroxybenzoate,
propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.
The pharmaceutical composition of the present disclosure may
further contain a lubricant, a wetting agent, a sweetening agent, a
flavoring agent, an emulsifying agent, a suspending agent, a
preservative, and the like, in addition to the above components.
Suitable pharmaceutically acceptable carriers and formulations are
described in detail in Remington's Pharmaceutical Sciences
(19.sup.th ed., 1995).
[0089] The vaccine composition and pharmaceutical composition of
the present disclosure may be administered orally or parenterally,
preferably parenterally. For example, the composition may be
administered by intravenous injection, local injection or
intraperitoneal injection.
[0090] The appropriate dosage of each of the vaccine composition
and the pharmaceutical composition of the present disclosure varies
depending on factors such as the formulation method, the mode of
administration, the patient's age, body weight, sex and
pathological condition, diet, the duration of administration, the
route of administration, excretion rate, and response sensitivity,
and an ordinarily skilled physician can easily determine and
prescribe an effective dosage for desired treatment or prevention.
According to a preferred embodiment of the present disclosure, the
daily dose of each of the vaccine composition and the
pharmaceutical composition of the present disclosure is 0.0001 to
100 mg/kg.
[0091] The vaccine composition and pharmaceutical composition of
the present disclosure may be formulated in unit dosage forms or in
multi-dosage packages using a pharmaceutically acceptable carrier
and/or excipient according to a method that may be easily carried
out by those skilled in the art. At this time, the formulation may
be a solution, suspension or emulsion in oil or aqueous medium, or
an extract, powder, granule, tablet or capsule, and may further
contain a dispersing agent or a stabilizer.
[0092] Each of the vaccine composition and pharmaceutical
composition of the present disclosure may be used as a single
therapy, but may also be used in combination with other
conventional chemotherapy or radiotherapy. When this combination
therapy is performed, cancer treatment may be more effectively
achieved. Chemotherapeutic agents that may be used together with
the composition of the present disclosure include cisplatin,
carboplatin, procarbazine, mechlorethamine, cyclophosphamide,
ifosfamide, melphalan, chlorambucil, bisulfan, nitrosourea,
dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin,
mitomycin, etoposide, tamoxifen, taxol, transplatinum,
5-fluorouracil, vincristin, vinblastine, and methotrexate.
Radiotherapies that may be used together with the composition of
the present disclosure include X-ray radiation and .gamma.-ray
radiation.
[0093] According to another aspect of the present disclosure, the
present disclosure provides a method of providing information for
diagnosis of a disease caused by overexpression of EPB41L5.
[0094] In the present disclosure, the method comprises steps of:
(a) obtaining a sample isolated ex-vivo from a subject; (b)
treating the sample with the monoclonal antibody or fragment
thereof and (c) determining whether the expression level of EPB41L5
contained in the sample from the subject is higher than the
expression level of EPB41L5 contained in a normal sample.
[0095] In the method of providing information according to the
present disclosure, contents related to the disease caused by
overexpression of EPB41L5, cancer, cancer stem cells, monoclonal
antibody, etc., are the same as described above, and thus
description thereof is omitted to avoid excessive complexity of the
specification.
[0096] According to still another aspect of the present disclosure,
the present disclosure provides a method for quantifying the amount
of EPB41L5 contained in a sample, the method comprising a step of
treating the sample with the monoclonal antibody or fragment
thereof.
[0097] Since the monoclonal antibody or fragment thereof according
to the present disclosure binds specifically to EPB41L5, the use
thereof makes it possible to accurately measure the amount of
EPB41L5 contained in the sample.
[0098] In the method for quantifying the amount according to the
present disclosure, contents related to the disease caused by
overexpression of EPB41L5, cancer, cancer stem cells, monoclonal
antibody, etc., are the same as described above, and thus
description thereof is omitted to avoid excessive complexity of the
specification.
[0099] According to yet another aspect of the present disclosure,
the present disclosure provides a kit for quantifying the amount of
EPB41L5, the kit comprising the monoclonal antibody or fragment
thereof
[0100] The kit for quantifying the amount according to the present
disclosure may quantify the amount of EPB41L5 by analyzing the
antigen against the antibody through an antigen-antibody binding
reaction. The antigen-antibody binding reaction is preferably
performed by one selected from the group consisting of conventional
ELISA (enzyme-linked immunosorbent assay), RIA (radioimmnoassay),
sandwich assay,
[0101] Western blotting on polyacrylamide gel, immunoblot assay,
and immunohistochemical staining, but is not limited thereto.
[0102] A support that is used for the antigen-antibody binding
reaction of the present disclosure may be selected from the group
consisting of a nitrocellulose membrane, a PVDF membrane, a well
plate synthesized using polyvinyl resin or polystyrene resin, and
slide glass, but is not limited thereto.
[0103] The secondary antibody is preferably labeled with a
conventional coloring agent for a color development reaction.
Specifically, it is possible to use any one labeling agent selected
from the group consisting of fluoresceins such as HRP (horseradish
peroxidase), alkaline phosphatase, colloidal gold, FITC (poly
L-lysine-fluorescein isothiocyanate or RITC
(rhodamine-B-isothiocyanate), and dyes. A substrate for inducing
color development is preferably chosen depending on the labeling
agent for color development reaction. Specifically, the substrate
is preferably any one selected from the group consisting of TMB
(3,3',5,5'-tetramethylbenzidine), ABTS
[2,2'-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid)] and OPD
(o-phenylenediamine), but is not limited.
Advantageous Effects
[0104] The features and advantages of the present disclosure are
summarized as follows:
[0105] (1) The present disclosure provides a monoclonal antibody
and a fragment thereof that recognizes EPB41L5 as an antigen and
binds specifically thereto.
[0106] (2) The present disclosure may be used to identify an
antibody that binds specifically to an epitope of EPB41L5, and an
antibody identified through this method functions to inhibit
EPB41L5 signaling and, and this function enables the antibody to be
effectively used as a vaccine or therapeutic agent against cancer
in which EPB41L5 is involved, particularly gastric cancer.
BRIEF DESCRIPTION OF DRAWINGS
[0107] FIG. 1A shows the results of performing microarray analysis
in gastric cancer patient specimens, which indicate that, when the
expression level of EPB41L5 is high, the prognosis of gastric
cancer patients is poor. FIG. 1A shows the results of performing
oligonucleotide microarray analysis in gastric cancer patients
without lymph node metastasis. The gastric cancer patients were
classified into two groups according to their gene expression
patterns (based on the median expression level of EPB41L5). FIG. 1B
is a graph showing the results of analyzing the 10-year survival
rates of two groups of gastric cancer patients. FIG. 1C shows the
results of performing Kaplan Meier plot analysis on two groups of
gastric cancer patients.
[0108] FIGS. 2A-2E show the data of various experiments performed
to analyze whether EPB41L5 and mesenchymal gene expression has
relevance with TGF.beta. signaling. FIG. 2A shows the results of
analyzing various gastric cancer cells by Western blotting.
[0109] FIG. 2B is a graph showing the results of analyzing the
expression level of EPB41L5 gene by performing real-time qPCR on
gastric cancer cells treated with TGF-.beta.1 (10 ng/ml) for 24
hours and on gastric cancer cells not treated cells with
TGF-.beta.1. Here, control indicates EPB41L5 mRNA expression in
gastric cancer cells not treated with TGF-.beta.1, and TGF-.beta.1
indicates EPB41L5 mRNA expression in gastric cancer cells treated
with TGF-.beta.1.
[0110] FIG. 2C shows the results of analyzing the expression levels
of EMT-related genes (EPB41L5, p-Smad2, Smad2, p-Smad3, Smad3,
PAI-1, and .beta.-actin) by performing Western blot analysis on
gastric cancer cells treated with TGF-.beta.1 (10 ng/ml) for 24
hours and on gastric cancer cells not treated cells with
TGF-.beta.1.
[0111] FIG. 2D shows the results of analyzing the expression levels
of endogenous EPB41L5 and E-cadherin in NCI-N87 cells and KATOIII
cells treated with TGF-.beta.1 (10 ng/ml) for 24 hours and in
NCI-N87 cells and KATOIII cells (control) not treated with
TGF-.beta.1. Here, green represents EPB41L5, red represents
E-cadherin, blue represents Hochest 33258, and scale bars represent
20 .mu.m.
[0112] FIG. 2E depicts images showing the cell morphologies of
KATOIII and SNU719 cells treated or not treated with TGF-.beta.1.
The cell morphology according to the presence or absence of
TGF-.beta.1 was observed through FIG. 2E. White arrows indicate
cells transformed to mesenchymal cells. Exposed KATOIII and SNU719
cells. Scale bars represent 20 .mu.m.
[0113] FIGS. 3A-3D show the results of an experiment performed to
confirm that Smad-depending TGF.beta. signaling may regulate
EPB41L5 expression. FIG. 3A shows the results of performing Western
blot analysis after MKN45 cells and NCI-N87 cells deficient in
Smad4 expression were treated or not treated with TGF-.beta.1.
[0114] FIG. 3B shows the results of immunofluorescent staining
analysis performed to analyze the expression levels of endogenous
EPB41L5 and E-cadherin in NCI-N87 cells treated or not treated with
TGF-.beta.1. Here, green represents EPB41L5, red represents
E-cadherin, and scale bars represent 20 .mu.m.
[0115] FIG. 3C is a graph showing the results of analyzing the mRNA
(fold induction) of each of Smad4, EPB41L5, Slug and PAI-1 in
KATOIII cells treated or not treated with TGF-.beta.1 or siSMAD4#1
or #2. Experimental results are expressed as mean.+-.s.e.m, and
scale bars represent 20 .mu.m. *P<0.05, **P<0.01,
***P<0.001.
[0116] FIG. 3D shows the results of analyzing the expression levels
of EMT-related genes (EPB41L5, p-Smad4, .alpha.PAI-1, .alpha.Slug,
and .alpha.-tubulin) by performing Western blot analysis on KATOIII
cells treated with TGF-.beta.1 (10 ng/ml) or siSmad4#1 or #2 and on
untreated KATOIII cells.
[0117] FIG. 4 shows the results of immunofluorescent staining
performed to analyze expression of endogenous EPB41L5 and
E-cadherin in untreated KATOIII cells, KATOIII cells treated with
only TGF-.beta.1, and KATOIII cells treated with TGF-.beta.1 and
then with a TGF.beta. inhibitor (LY2157299). Green represents
EPB41L5, red represents E-cadherin, blue represents Hochest 33258,
and scale bars represent 20 .mu.m.
[0118] FIG. 5A shows the results of performing real-time qPCR on NC
siRNA-transfected KATOIII cells, and EPB41L5 siRNA #1- or EPB41L5
siRNA #2-transfected KATOIII cells depending on the presence or
absence of TGF-.beta.1 in order to confirm that the migration of
gastric cancer cells caused by EPB41L5 is regulated by treatment
with TGF-.beta.1.
[0119] FIG. 5B shows the results of performing Western blot
analysis on NC siRNA-transfected KATOIII cells, and EPB41L5 siRNA
#1- or EPB41L5 siRNA #2-transfected KATOIII cells depending on the
presence or absence of TGF-.beta.1.
[0120] FIG. 5C shows the results of measuring morphological changes
of NC siRNA-transfected KATOIII cells, EPB41L5 siRNA #1- or EPB41L5
siRNA #2-transfected KATOIII cells and SNU719 cells depending on
the presence or absence of TGF-.beta.1. White arrows indicate cells
transformed to mesenchymal cells, and scale bars represent 20
.mu.m.
[0121] FIG. 5D depicts images showing the results of performing in
vitro migration and invasion analysis on NC siRNA-transfected
KATOIII cells and EPB41L5 siRNA #1- or EPB41L5 siRNA #2-transfected
KATOIII cells depending on the presence or absence of TGF-.beta.1.
Scale bars represent 20 .mu.m.
[0122] FIG. 5E shows the results of performing in vitro migration
and invasion analysis on NC (negative control) siRNA-transfected
KATOIII cells and EPB41L5 siRNA #1- or EPB41L5 siRNA #2-transfected
KATOIII cells depending on the presence or absence of TGF-.beta.1.
Experimental results are expressed as mean.+-.s.e.m. *P<0.05,
**P<0.01, ***P<0.001.
[0123] FIGS. 6A-6H relate to the specificity of an anti-EPB41L5
monoclonal antibody. FIG. 6A shows the results of performing
Western blot analysis on MKN28 cells transfected with a
Flag-EPB41L5 construct, and FIG. 6B shows the results of performing
Western blot analysis on MKN28 cells transfected with EPB41L5 siRNA
(#1 or #2).
[0124] FIG. 6C shows the results of performing immunofluorescent
staining of EPB41L5 siRNA-transfected MKN28 cells (EPB41L5 siRNA).
Here, green represents EPB41L5.
[0125] FIG. 6D is a schematic view showing the structure of an
EPB41L5 domain.
[0126] FIG. 6E shows the results of performing Western blot
analysis on 293T cells transfected with each of the FL (full
length) of EPB41L5, the FERM of EPB41L5 and the C-terminus of
EPB41L5.
[0127] FIG. 6F shows the results of analyzing Western blot analysis
on total lysates of 293T cells transfected with plasmids (1-633,
1-628, 1-623, 1-618, and 1-613) in which five amino acid residues
at the C-terminus of EPB41L5 are continuously deleted.
[0128] FIG. 6G shows the results of analyzing Western blot analysis
on total lysates of 293T cells transfected with plasmids consisting
of FL (full length) of EPB41L5 and .DELTA.619-624 of EPB41L5,
respectively.
[0129] FIG. 6H shows the results of performing immunofluorescent
staining of MKN28 cells co-transfected with EPB41L5 siRNA and any
one selected from among FL (full length) of EPB41L5, FERM of
EPB41L5, the C-terminus of EPB41L5, and .DELTA.619-624 of EPB41L5.
At this time, the immunofluorescent staining was performed using
anti-EPB41L5 mAb and anti-Flag antibodies. Green represents
EPB41L5, red represents Flag tag, blue represents Hochest, and
scale bars represent 20 .mu.m.
[0130] FIG. 7A depicts cell migration images and FIG. 7B depicts an
analysis result graph, obtained by treating KATOIII cells with
various concentrations of anti-EPB41L5 mAb depending on the
presence or absence of TGF-.beta.1 in order to confirm that the
invasion and metastasis of gastric cancer cells that was increased
by TGF-.beta.1 is inhibited by an anti-EPB41L5 monoclonal antibody.
Experimental data are expressed as mean.+-.s.e.m, and scale bars
represent 20 .mu.m. **P<0.01 and ***P<0.001.
[0131] FIG. 8 shows the results of comparing the degree to which
the invasion and metastasis of gastric cancer cells that was
increased by TGF-.beta.1 is inhibited by an anti-EPB41L5 monoclonal
antibody, between the monoclonal antibody of the present disclosure
and an antibody that recognizes other epitopes.
[0132] FIGS. 9A-9D show the results of an experiment performed to
confirm the relevance of EPB41L5 with the lung metastasis of
gastric cancer cells. FIG. 9A shows the results of performing in
vitro migration and invasion analysis on EPB41L5-overexpressing
KATOIII cells and control cells (vehicle). At this time, 1% FBS was
used as a chemoattractant.
[0133] FIG. 9B shows quantification of the results of performing in
vitro migration and invasion analysis on EPB41L5-overexpressing
KATOIII cells and control cells (vehicle). 15 random cells were
selected from each group and quantified by cell counting using
Fusion Capt advanced software. Experimental data are expressed as
mean.+-.s.e.m.
[0134] FIG. 9C shows the results obtained by injecting
EPB41L5-overexpressing KATOIII cells (GFP-labeled) (EPB41L5(GFP))
into nude mice through the tail vein (10 mice per group) and
analyzing lung metastasis of the cells using an IVIS optical
imaging system. FIG. 9D shows the results of quantifying the
intensity, measured from the images of FIG. 9C, using an ROI tool.
Experimental data are expressed as average radiant
efficiency.+-.s.e.m. *P<0.05 and * P<0.01.
[0135] FIG. 10A is a diagram showing that a (EPB41L5+mAb) group is
prepared by injecting EPB41L5-overexpressing KATOIII cells into
nude mice through the tail vein and injecting an anti-EPB41L5
monoclonal antibody into the mice at a dose of 5 mg/kg once every 2
days for 2 weeks.
[0136] FIG. 10B shows the results of analyzing a control group
(vehicle), an (EPB41L5) group obtained by injecting
EPB41L5-overexpressing KATOIII cells into nude mice through the
tail vein, and an (EPB41L5+mAb) group obtained by injecting
EPB41L5-overexpressing KATO.sup.2 cells into nude mice through the
tail vein and injecting an anti-EPB41L5 monoclonal antibody into
the mice at a dose of 5 mg/ml once every 2 days for 2 weeks, by an
IVIS optical imaging system.
[0137] FIG. 10C is a graph showing the results of quantifying
intensity from the images of FIG. 9B by an ROI tool. Experimental
data are expressed as average radiant efficiency.+-.s.e.m.
**P<0.01.
[0138] FIG. 11 shows the results of analyzing the expression and
antibody function of EBP41L5 protein in gastric cancer, lung cancer
and breast cancer cells by Western blot analysis using an EPB41L5
antibody.
[0139] FIG. 12 is a graph showing the results of analyzing the cell
viabilities of gastric cancer, lung cancer and breast cancer cells
treated with various concentrations of an EBP41L5 antibody.
BEST MODE
[0140] According to one embodiment of the present disclosure, there
are provided: an epitope comprising the 619.sup.th to 624.sup.th
amino acid residues of an EPB41L5 protein represented by the amino
acid sequence of SEQ ID NO: 1; and a vaccine composition for
preventing or treating cancer containing the same.
[0141] According to another embodiment of the present disclosure,
there is provided a monoclonal antibody or fragment thereof
comprising: a heavy-chain variable region comprising a heavy-chain
CDR1 represented by SEQ ID NO: 6, a heavy-chain CDR2 represented by
SEQ ID NO: 7, and a heavy-chain CDR3 represented by SEQ ID NO: 8;
and a light-chain variable region comprising a light-chain CDR1
represented by SEQ ID NO: 9, a light-chain CDR2 represented by SEQ
ID NO: 10, and a light-chain CDR3 represented by SEQ ID NO: 11.
[0142] According to still another embodiment of the present
disclosure, there is provided a composition for preventing or
treating cancer containing the monoclonal antibody or fragment
thereof as an active ingredient.
Mode for Invention
[0143] Hereinafter, the present disclosure will be described in
more detail with reference to examples. It will be obvious to those
skilled in the art that these examples serve to merely describe the
present disclosure in more detail, and the scope of the present
disclosure according to the subject matter of the present
disclosure is not limited by these examples.
Experimental Methods
1. Patient Specimens
[0144] Gastric cancer patient specimens for oligonucleotide
microarray and survival rate analysis were obtained from the
Clinical Test Center, Severance Hospital (IRB No: 4-2016-0013).
2. Cell Culture and Reagents
[0145] Human gastric cancer cell lines AGS, NCI-N87, KATO.sup.2,
SK-GT-4, MKN1, MKN28, MKN45, SNU1, SNUS, SNU16, SNU216, SNU484,
SNU638, SNU668 and SNU719 were obtained from professor Cheong
Jae-Ho lab at Yonsei University (Seoul, Korea).
[0146] All the gastric cancer cells were cultured in RPMI-1640
medium supplemented with 10% FBS and 1% antibiotic/antimycotic
solution (Corning, Manassas, Va., USA)) at 37.degree. C. under 5%
CO.sub.2. 293FT cells were cultured in DMEM. TGF-.beta.1 was
purchased from Prospec (East Brunswick, N.J., USA), and LY2157299
was purchased from Selleckchem (Houston, Tex., USA).
3. Plasmids
[0147] EPB41L5-relevant DNA constructs encoding full-length, FERM
domain, C-terminus, .DELTA.619-624 and C-terminal 5 amino acids,
which were sequentially removed, were generated by PCR and cloned
into the pSG5-KF2M1-Flag (Sigma-Aldrich, St. Louis, Mo., USA). All
plasmid constructs were verified by DNA sequencing.
4. siRNA Transfection
[0148] Cells were transfected with siRNAs using Lipofectamine
RNAiMAX (Thermo Fisher Scientific, Rockford, Ill., USA) according
to the manufacturer's procedure. siRNAs used were synthesised by
Genolution (Seoul, South Korea) and had the following siRNA
sequences: EPB41L5-1 (EPB41L5 siRNA #1; siEPB41L5#1) (5'-GC
[0149] AAUUGGCAGCUUAUAAUUU-3'), EPB41L5-2 (EPB41L5 siRNA #2;
siEPB41L5#2) (5'-UUCAGAUUC GUGCCUAUUCAGUU-3'), Smad4-1
(5'-GCUACUUACCAUCAU AACAUU-3'), and Smad4-2
(5'-GUUCCAUUGCUUACUUUUUUUUU-3').
5. Monoclonal Antibody)_Anti-EPB41L5 mAb
[0150] Monoclonal antibody was produced by immunizing mice with the
386.sup.thto 637.sup.th amino acid residues (SEQ ID NO: 3) of human
EPB41L5 antigen, and this production was carried out by ATgen
(Seongnam, Korea).
1) Immunization of Mice and Production of Hybridoma Cells
[0151] Monoclonal antibody was produced using the 386.sup.th to
637.sup.th amino acid residues (SEQ ID NO: 3) of human EPB41L5
antigen. For efficient production of the antibody, SEQ ID NO: 3 was
selected as a target peptide for antibody production.
[0152] The target peptide for antibody production (SEQ ID NO: 3)
was injected into mice, and cells producing the antibody against
the antigen were generated. The antibody-producing cells (B
lymphocytes) obtained from the spleen of the mice were fused with
myeloma cells to obtain hybridoma cells. The hybridoma cells were
cultured in HAT medium in which only hybridoma cells can survive,
and then the activity of the antibody was analyzed by ELISA
assay.
2) Selection and Identification
[0153] Among the hybridoma cells, monoclonal hybridoma cells that
specifically recognize EPB41L5 were selected, and the selected
monoclonal hybridoma cells were injected into the abdominal cavity
of mice, and then monoclonal antibody was recovered from ascites.
The monoclonal antibody of the present disclosure, recovered from
ascites, was purified using protein A and protein G columns.
[0154] Thereafter, a monoclonal antibody (EPB41L5 mAb) that
specifically recognizes EPB41L5 was selected and identified by
Western blot analysis and immunofluorescent staining using the
EPB41L5 construct and siRNA.
6. Generation of EPB41L5-Overexpressing Stable Cell Lines
[0155] EPB41L5 DNA was cloned into the pCDH-CMV-MCS-EF1-puro vector
(Addgene, Cambridge, Mass., USA). To generate lentiviral particles,
packaging plasmids pRSV-Rev and pMD2.G were transfected with
pCDH-EPB41L5 or pLECE3-GFP in 293FT cells. After 48 hours of
incubation, the supernatants were collected and filtered with a
0.45-.mu.m pore filter. The KATO.sup.2 cells were infected with
lentiviral particles and then selected with 1 .mu.g/ml puromycin
(Sigma-Aldrich). The selected stable EPB41L5-overexpressing cells
were sorted with GFP by an Aria II flow cytometer (BD Biosciences,
Sparks, Md., USA).
7. Western Blot Analysis
[0156] Cells were lysed in lysis buffer (20 mmol/L Tris-Cl, 150
mmol/L NaCl, 1% Triton X-100, 1.5% MgCl.sub.2, 1 mMEDTA, 1 mM
Na.sub.2VO.sub.4, 1 mM phenylmethylsulfonyl fluoride (PMSF), and
protease inhibitor cocktail, pH 7.5). The lysates were vortexed
briefly and centrifuged at 13,000 rpm for 20 minutes at 4.degree.
C. The supernatants were collected and transferred into fresh
tubes. Protein concentrations were measured at 660 nm using protein
assay reagent (Thermo Fisher Scientific). Equal concentrations of
protein samples were prepared, and each sample was electrophoresed
on SDS-polyacrylamide gel and then transferred to nitrocellulose
membranes (Whatman, Dassel, Germany). The membranes were blocked in
Tris-buffer (pH 7.4) containing 0.1% (v/v) Tween 20 (Sigma-Aldrich)
and 5% (w/v) nonfat Difco.TM. skim milk (BD Biosciences) and probed
with primary antibodies. The following antibodies were used:
polyclonal EPB41L5 (Thermo Fisher Scientific); EPB41L5 (ATGen,
Seongnam, Korea); Flag-tag, .beta.-actin (Sigma-Aldrich);
.alpha.-tubulin (Abcam, Cambridge, GB), WI, T.beta.RII, Smad2,
Smad3, Smad4, p-Smad2, p-Smad3, Slug, ZEB1 (Cell signaling,
Danvers, Mass., USA); and PAI-1(Santa Cruz, Dallas, Tex., USA). The
signals were developed by substrate (Thermo Fisher Scientific)
according to the manufacturer's protocol.
8. RNA Isolation and Real-Time qPCR
[0157] Total RNA was extracted using TRIzol reagent (Takara Bio,
Otsu, Shiga, Japan), and cDNA was synthesised using PrimeScript
Reverse Transcriptase (Takara Bio, Otsu, Shiga, Japan) and
oligo(dT) according to the manufacturer's protocol. quantitative
PCR (qPCR) was performed using SYBR Green Master (Roche, Basel,
Switzerland) and ABI Prism 7700 sequence detection systems (Applied
Biosystems, Carlsbad, Calif., USA). The following primers were used
to detect transcripts: .alpha.-tubulin (5'-TTCTCCATTTACCCGGCACC-3')
and (5'-GTTAGTGTAGGTTGGGCGCT-3'); EPB41L5 (5'-GAAA
GAAGGCCCAGCAAACG-3') and (5'-AGATCTCATCCCCCAAGCCT-3'); PAI-1
(5'-CCCCACTTCTTCAGGCTGTT-3') and (5'-GCCGTTGAA GTAGAGGGCAT-3');
Slug (5'-TCATCTTTGGGGCGAGTGAG-3') and (5'-TGCAGCTGCTTATGTTTGGC-3');
ZEB 1 TATGAATGCCCAAACTGCAA-3') and (5'-TGGTGATGCTGAAAGAGACG-3');
Smad4 (5'-TGCATGACTTTGAGGGACAG-3') and
(5'-GTGGAAGCCACAGGAATGTT-3'). The quantity of cDNA was normalized
with .alpha.-tubulin. All experiments were performed in triplicate,
and relative expression levels and standard deviations were
calculated by the comparative method.
9. Immunofluorescence Analysis
[0158] Cells were cultured on chamber slides (SPL Life Sciences,
Pocheon-shi, Korea) and fixed in 4% paraformaldehyde for 30 minutes
at room temperature. After washing with PBS, the fixed cells were
incubated for 1 hour with 3% BSA to block nonspecific antibodies.
Anti-EPB41L5, anti-E-cadherin and anti-Flag-tag were incubated at
4.degree. C. overnight and then stained with Alexa Fluor 488- or
Alexa Fluor 549-conjugated goat anti-rabbit or anti-mouse secondary
antibody (Thermo Fisher Scientific). The nuclei were stained with
Hoechst 33258. The samples were imaged with an LSM710 confocal
microscope (Carl Zeiss, Oberkochen, Germany).
10. In Vitro Migration and Invasion Assay
[0159] Cell migration was measured by Transwell with 8.0-.mu.m pore
polycarbonate membrane insert (Corning, Manassas, Va., USA). For
invasion assay, inserts of the Transwell were coated with Matrigel
(BD Biosciences). 1.times.10.sup.5 cells per well were added to the
upper chamber, and the lower chamber was filled with 600 .mu.L of
serum-free medium with or without TGF.beta.1, LY2157299 (TGF.beta.
inhibitor), or anti-EPB41L5 monoclonal antibody (mAb) as a
chemoattractant. After 24 hours of incubation, nonmigrating or
noninvading cells were carefully removed from the upper chamber by
a cotton swab. Migrating or invading cells were stained with 0.2%
crystal violet (Sigma-Aldrich) in 20% methanol and counted at
.times.200 magnification under a microscope. The migration and
invasion assays were performed in triplicate.
11. In Vivo Metastasis Assay
[0160] 5-week-old athymic BALB/c nu/nu mice were obtained from
Orient (Seoul, Korea). Control vector or pCDH-EPB41L5- and
pLECE3-GFP-overexpressing KATO.sup.2 cells (2.times.10.sup.7 cells
in 200 .mu.L PBS solution) were injected into the lateral tail
vein. Anti-EPB41L5 monoclonal antibody was administered at 5 mg/kg
every day for 2 weeks. The fluorescence images were taken and
analyzed with an IVIS imaging system (Caliper Life Sciences,
Hopkinton, Mass., USA).
12. Statistical Analysis
[0161] The overall survival rate in the data obtained from gastric
cancer patient specimens was analyzed using the Kaplan-Meier
Plotter (http://kmplot.com/analysis). Used Affymetrix ID
was220977_x_at, and the log-rank test was used for Kaplan-Meier
survival plots. Statistical analysis was performed using the
Student t test to compare two groups of independent experiments
(two sided). The data were expressed as mean.+-.standard deviation
(SD). P values<0.05 were considered statistically
significant.
EXAMPLES
<Example 1>. Analysis of Expression Level of EPB41L5 Gene in
Gastric Cancer Patient Specimens
[0162] To identify potential target molecules in GC,
oligonucleotide microarray analysis was performed in 78 gastric
cancer patients without lymph node metastasis. The patients were
divided into two groups (first and second groups) based on the
median expression level of EPB41L5 gene. With respect to the median
expression level of EPB41L5 gene, the first group is positioned on
the left side and the second group is positioned on the right
side.
[0163] It was confirmed that the first group with highly expressed
APEG1, SMPX, GPR177 and EPB41L5 genes had a higher rate of
recurrence or death than the second group in which the expression
levels of the genes were not high (FIG. 1A). Among the APEG1, SMPX,
GPR177 and EPB41L5 genes, EPB41L5 was selected as a target protein
for antibody-based cancer therapy. As a result of analyzing the
future 10-year survival rate of the two groups divided based on the
median expression level of the EPB41L5 gene (FIG. 1B), it can be
seen that the survival rate of the patients with high expression
levels of the EPB41L5 gene is lower than that of the patients with
low expression levels of the EPB41L5 gene.
[0164] In addition, as a result of performing Kaplan Meier plot
analysis on the two groups of cancer patients (876 gastric cancer
patients), it was confirmed that, in the Kaplan Meier plotter using
a database such as GEO, EGA or TCGA, gastric cancer patients with
high levels of EPB41L5 expression had a lower survival rate (HR
1.73, P 2.8E-10) (FIG. 1C). These clinical outcomes show that high
levels of EPB41L5 expression are related to poor prognosis in
gastric cancer patients. In other words, gastric cancer patients
with high levels of EPB41L5 gene expression had a lower survival
rate.
<Example 2>. Analysis of Expression Patterns of TGF-.beta.1
and EPB41L5 Proteins in Gastric Cancer Cells
[0165] The present inventors examined which gastric cancer cells
were responsive to TGF.beta. signaling. The protein levels of
proteins relevant to TGF.beta. signaling cascades were analyzed by
performing Western blot analysis in several gastric cancer cell
lines (FIG. 2A). As a result, it was confirmed that Smad3 or Smad4
was not detected in NCI-N87, MKN45, SNU216 and SNU484 cells.
[0166] Next, gastric cancer cells were treated with TGF-.beta.1 to
examine whether EPB41L5 gene expression is regulated by TGF.beta.
signaling. As a result, it was confirmed that the expression level
of the EPB41L5 gene was significantly increased by TGF-.beta.1
treatment in KATOIII, MKN28, SNU1 and SNU719 cells, but not in
Smad3/Smad4-defective GC cells (FIG. 2B). In addition, expression
of mesenchymal genes such as PAI-1, Slug and phosphorylated Smad2
and Smad3 was significantly increased by TGF-.beta.1 treatment
(FIG. 2C).
[0167] According to the results of immunofluorescence staining
analysis (FIG. 2D), it was confirmed that expression of the
epithelial marker E-cadherin in the transmembrane decreased and the
expression level of EPB41L5 protein was increased by TGF-.beta.1 in
the cell membrane of KATOIII. In addition, the morphologic
characteristics of gastric cancer cells KATOIII and SNU719 were
changed to mesenchymal characteristics by TGF.beta.1 treatment
(FIGS. 2E and 2F).
[0168] These results suggest that TGF.beta. signaling regulates the
expression levels of the EPB41L5 gene and the protein encoded
thereby and epithelial-mesenchymal transition in cancer cells, for
example, gastric cancer cells.
<Example 3>. Regulation of Expression Level of EPB41L5
Protein by Smad-Dependent TGF.beta. Signaling
[0169] To examine whether the TGF.beta.1-induced increase in the
expression level of the EPB41L5 protein is Smad-dependent, Western
blot analysis and immunofluorescent staining were performed in
Smad4-deficient MKN45 (Smad4-knocked down MKN45) and NCI-N87 cells
(gastric cancer). It was confirmed that the levels of these genes
and the EPB41L5 proteins in each type of cells treated with
TGF-.beta.1 did not change (FIGS. 3A and 3B). In addition, it was
confirmed that EPB41L5, PAI-1 and Slug in each type of cells
treated with TGF-.beta.1 were unregulated, and expression of
EPB41L5, PAI-1 and Slug in MKN28 cells, in which Smad4 was knocked
down using siRNA, was inhibited (FIGS. 3C and 3D).
[0170] Taking these results together, it can be seen that TGF.beta.
signaling is Smad-dependent, indicating that TGF.beta. signaling is
involved in the regulation of EPB41L5 expression in gastric cancer
cells.
<Example 4>. Analysis of Relevance of TGF-.beta.1 with
EPB41L5 Protein
[0171] In the present disclosure, changes in the expression level
of the EPB41L5 protein by treatment with the potent TGF.beta.
receptor I inhibitor LY2157299 (Galunisertib) were analyzed.
[0172] FIG. 4 shows the results of immunofluorescent staining
performed to analyze expression of endogenous EPB41L5 and
E-cadherin in untreated KATOIII cells, KATOIII cells treated with
only TGF-.beta.1, and KATOIII cells treated with TGF-.beta.1 and
then with a TGF.beta. inhibitor (LY2157299). Green represents
EPB41L5, red represents E-cadherin, blue represents Hochest 33258,
and scale bars represent 20 .mu.m.
[0173] As shown in FIG. 4, as a result of performing
immunofluorescent staining of KATOIII cells treated with only
TGF-.beta.1 and KATOIII cells treated sequentially with TGF-.beta.1
and LY2157299, it was confirmed that the expression level of the
EPB41L5 protein was increased by TGF-.beta.1, but was inhibited by
LY2157299.
<Example 5>. Analysis of Effect of EPB41L5 on Migration of
Gastric Cancer Cells
[0174] Whether EPB41L5 affects the migration of gastric cancer
cells in response to TGF.beta. signaling was examined. To this end,
KATOIII cells or SNU719 cells were transfected with EPB41L5 siRNA.
Specifically, two types of siRNAs (EPB41L5 siRNA #1 (SEQ ID NO 4:
5'-GC AAUUGGCAGCUUAUAAUUU-3'), and EPB41L5 siRNA #2 (SEQ ID NO 5:
5'-UUCAGAUUC GUGCCUAUUCAGUU-3')) were used for knockdown of
EPB41L5. It was confirmed that the gene and protein levels of
EPB41L5 and PAI-1 in the KATOIII cells transfected with EPB41L5
siRNA #1 or EPB41L5 siRNA #2 significantly decreased compared to
those in the KATOIII cells transfected with NC siRNA (FIGS. 5A and
5B). However, in the KATOIII cells transfected with EPB41L5 siRNA
#1 or EPB41L5 siRNA #2, changes in phosphorylated Smad3
(.alpha.p-Smad3) and Smad3 (.alpha.Smad3) were not observed (FIG.
5B).
[0175] In addition, as a result of analyzing epithelial-mesenchymal
transition and the migration of gastric cancer cells when the
KATOIII cells transfected with NC siRNA and the KATOIII cells
transfected with EPB41L5 siRNA #1 or EPB41L5 siRNA #2 were treated
with TGF-.beta.1, it was confirmed that knockdown of EPB41L5
blocked the effect of TGF-.beta.1 on epithelial-mesenchymal
transition and the migration of gastric cancer cells (FIGS. 5C, 5D
and 5E). That is, it can be seen that EPB41L5 is an essential
element for cell metastasis and gastric cancer cell migration which
are induced by TGF.beta..
<Example 6>. Anti-EPB41L5 mAb (Monoclonal Antibody)
Recognizes 619-624 AA Sequence of C-Terminus
[0176] In the present disclosure, it was considered that, since
cell adhesion molecules play an important role in metastasis, it is
possible to develop new therapeutic agents using monoclonal
antibodies and peptides. Thus, a monoclonal antibody (mAb) against
EPB41L5 was developed.
[0177] To validate the specificity of the developed antibody,
gastric cancer cells transfected with Flag-EPB41L5 or EPB41L5 siRNA
were analyzed by Western blot analysis and immunofluorescence
analysis. As a result, it was confirmed that overexpressed EPB41L5
was detected in the gastric cancer cells transfected with
Flag-EPB41L5 (FIG. 6A), and that EPB41L5 was silenced in the
gastric cancer cells (NC, siRNA #1 or siRNA #2) transfected with
EPB41L5 siRNA (FIG. 6B). In addition, as a result of analyzing the
EPB41L5 siRNA-transfected gastric cancer cells by immunofluorescent
staining, it can be seen that EPB41L5 was apparently detected by
the anti-EPB41L5 monoclonal antibody in the gastric cancer cells
(FIG. 6C). As a result of analyzing the anti-EPB41L5 monoclonal
antibody in the present disclosure by Western blot analysis, it was
confirmed that the anti-EPB41L5 monoclonal antibody recognized the
C-terminus of EPB41L5, not the N-terminal FERM domain of EPB41L5
(FIGS. 6D and 6#).
[0178] In addition, as a result of analyzing the antibodies in
which 5 amino acids at the C-terminus of EPB41L5 were sequentially
removed, it was confirmed that the antibodies recognized amino
acids 619-624 of the C-terminus of EPB41L5 (FIGS. 6F and 6G).
Considering the characteristics of nonpermeabilized
immunofluorescent staining, in order to inhibit expression of
endogenous EPB41L5, cells were co-transfected with EPB41L5 siRNA
and any one plasmid selected from among FL (full length) of
EPB41L5, FERM of EPB41L5, C-terminus of EPB41L5 and .DELTA.619-624
of EPB41L5. Then, expression of exogenous EPB41L5 was analyzed
using anti-Flag antibody. EPB41L5 expression was detected in FK
cells transfected with FL and C-terminal constructs and in MKN28
cells not transfected with A619-624 (FIG. 6H). Taking these results
together, it can be seen that the anti-EPB41L5 monoclonal antibody
specifically recognizes the 619-624 amino acid region of
EPB41L5.
<Example 7>. Sequencing of Variable Region of Anti-EPB41L5
Monoclonal Antibody
[0179] The sequence of the variable region of the monoclonal
antibody produced Example 6 above was analyzed by ATgen (Korea). As
a result, the sequences of the heavy-chain variable region and
light-chain variable region of the monoclonal antibody according to
the present disclosure are as follows:
TABLE-US-00001 Heavy-chain variable region (SEQ ID NO: 12)
QVQLKESGTVLARPGASVKMSCKASGYTFTSYWMHWVKQRPGQGLEW
IGAIYPGNSDTSYNQKFKDKAKLTAVTSTSTAYMELSSLTDEASAVY
YCTRGGKLPFAMDYWGQGTSVTVSS Light-chain variable region (SEQ ID NO:
13) DVLMTQTPLSLPVSLGDQASMSCRSSQSLVHSNGNTYLHWYLQKPGQ
SPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCS
QSTHVPWTFGGGTKLEIK
[0180] Furthermore, the sequences of the heavy-chain variable
region CDR1 to CDR3 and light-chain variable region CDR1 to CDR3 of
the monoclonal antibody according to the present disclosure are as
follows:
[0181] Heavy-chain variable region CDR1: Gly Tyr Thr Phe Thr Ser
Tyr Trp (SEQ ID NO: 6)
[0182] Heavy-chain variable region CDR2: Ile Tyr Pro Gly Asn Ser
Asp (SEQ ID NO: 7)
[0183] Heavy-chain variable region CDR3: Thr Arg Gly Gly Lys Leu
Pro Phe Ala Met Asp Tyr (SEQ ID NO: 8)
[0184] Light-chain variable region CDR1: Gln Ser Leu Val His Ser
Asn Gly Asn Thr Tyr (SEQ ID NO: 9)
[0185] Light-chain variable region CDR2: Lys Val Ser (SEQ ID NO:
10)
[0186] Light-chain variable region CDR3: Ser Gln Ser Thr His Val
Pro Trp Thr (SEQ ID NO: 11)
<Example 8>. Therapeutic Effect of Anti-EPB41L5 Monoclonal
Antibody
[0187] The specificity of the anti-EPB41L5 monoclonal antibody was
validated. In this experiment, as a result of evaluating the effect
of the anti-EPB41L5 monoclonal antibody on the migration of gastric
cancer (GC) cells with or without TGF-.beta.1 treatment, it was
confirmed that TGF-.beta.1 efficiently increased the migration of
KATOIII. Furthermore, it was confirmed that the anti-EPB41L5
monoclonal antibody inhibited the migration of KATOIII cells that
was increased by TGF-.beta.1 (FIGS. 7A and 7B).
<Example 9>. Comparison of Metastasis Inhibitory Effect of
Anti-EPB41L5 Monoclonal Antibody
[0188] In the same manner as in Example 8, the degree of cell
migration inhibited by the anti-EPB41L5 monoclonal antibody was
compared between different epitope sequences recognized by the
monoclonal antibody. As shown in FIG. 8, it was confirmed that the
monoclonal antibody (AtGen) according to the present disclosure
significantly (100%) inhibited cell migration that was increased by
TGF-.beta.1, whereas an antibody (Novus antibody) recognizing the
638.sup.th to 708.sup.th amino acid sequence of EPB41L5 protein
hardly inhibited cell migration that was increased by TGF-.beta.1.
Accordingly, it can be seen that the antibody recognizing the
619.sup.th to 624.sup.th amino acid sequence of the C-terminal
region of EPB41L5 can very effectively inhibit cell migration,
which was increased by TGF-.beta.1, compared to antibodies that
bind specifically to epitopes consisting of other amino acid
sequences.
<Example 10>. Analysis of Relevance of EPB41L5 with Gastric
Cancer Cell Metastasis
[0189] Through the above-described experiments, it was confirmed
that EPB41L5 expression, which was increased by TGF-.beta.1, is
involved in the in vivo migration and invasion of gastric cancer
cells. Accordingly, in the present disclosure, the effect of
EPB41L5 on the migration of gastric cancer cells was analyzed by in
vitro migration and invasion assay.
[0190] Specifically, EPB41L5-overexpressing KATOIII cells and
untreated KATOIII cells as a vehicle were prepared.
[0191] As a result of analyzing in vitro migration and invasion
assay in EPB41L5-verexpressing KATOIII cells, it was confirmed that
the migration and invasion of the EPB41L5-overexpressing KATOIII
cells substantially increased (FIGS. 9A and 9B). To observe the
effect of in vivo EPB41L5 expression on gastric cancer cells,
EPB41L5-overexpressing KATOIII cells were injected into nude mice
through the tail vein. As a result, it was confirmed that the lung
metastasis of gastric cancer cells in the EPB41L5(GFP) group
significantly increased compared to that in the vehicle(GFP) group
(FIGS. 9C and 9D). In summary, it can be seen that EPB41L5 promotes
in vivo metastasis of gastric cancer cells.
<Example 11>. Examination of Ability of Anti-EPB41L5
Monoclonal Antibody to Inhibit Metastasis of Gastric Cancer
Cells
[0192] The in vivo efficacy of the anti-EPB41L5 monoclonal antibody
was examined. To this end, the following groups were prepared: a
vehicle group; an (EPB41L5) group obtained by injecting
EPB41L5-overexpressing KATOIII cells into nude mice through the
tail vein; and an (EPB41L5+mAb) group obtained by injecting
EPB41L5-overexpressing KATOIII cells into nude mice through the
tail vein and 5 mg/kg of the anti-EPB41L5 monoclonal antibody into
the nude mice once every 2 days for 2 weeks (FIG. 10A).
[0193] It can be confirmed that, in the nude mice into which the
EPB41L5-overexpressing cells were injected, the lung metastasis of
cancer increased, but in the group into which the anti-EPB41L5
monoclonal antibody was injected, the lung metastasis of gastric
cancer cells decreased compared to that in the vehicle group (FIGS.
10B and 10C). Thereby, it can be seen that the anti-EPB41L5
monoclonal antibody according to the present disclosure exhibits
therapeutic or preventive effects against advanced gastric
cancer.
<Example 12>. Confirmation of Function of Anti-EPB41L5
Monoclonal Antibody That Specifically Recognizes EBP41L5
Protein
[0194] The specificity of the anti-EPB41L5 monoclonal antibody to
EBP41L5 was confirmed. To this end, KATOIII cells and MKN28 cells
were prepared as gastric cancer cell lines. As lung cancer cell
lines, A549 cells and H226 cells were prepared, and as breast
cancer cell lines, MCF7 cells and MDA-MB-231 cells were prepared.
The cells were obtained from Professor Cheong Jae-Ho lab at Yonsei
University and from the Korean Cell Line Bank. Each cell line was
cultured in RPMI-1640 medium supplemented with 10% FBS and 1%
antibiotic/antimycotic solution (Corning, Manassas, Va., USA)) at
37.degree. C. under 5% CO.sub.2.
[0195] Using the anti-EPB41L5 monoclonal antibody, EBP41L5 protein
expression in each cell group was analyzed by Western blot analysis
(FIG. 11). As a result, EBP41L5 protein expression was detected by
the anti-EPB41L5 monoclonal antibody not only in the gastric cancer
cell lines but also in the lung cancer cell lines and the breast
cancer cell lines (FIG. 11). This indicates that the anti-EPB41L5
monoclonal antibody according to the present disclosure may be
effectively used for treatment or prevention of not only gastric
cancer but also lung cancer and breast cancer.
<Example 11>. Examination of Cell-Specific Toxicity of
Anti-EPB41L5 Monoclonal Antibody
[0196] The effects of the anti-EPB41L5 monoclonal antibody of the
present disclosure on the cell viabilities of various cells were
examined. To this end, KATOIII cells and MKN28 cells were prepared
as gastric cancer cell lines. As lung cancer cell lines, A549 cells
and H226 cells were prepared, and as breast cancer cell lines, MCF7
cells and
[0197] MDA-MB-231 cells were prepared. The cells were obtained from
Professor Cheong Jae-Ho lab at Yonsei University and from the
Korean Cell Line Bank.
[0198] Each cell line was cultured in RPMI-1640 medium supplemented
with 10% FBS and 1% antibiotic/antimycotic solution (Corning,
Manassas, Va., USA)) at 37.degree. C. under 5% CO.sub.2.
[0199] 0, 2, 4 or 6 .mu.g of the anti-EPB41L5 monoclonal antibody
was injected into each cell group, and after 24 hours, the cell
viability (%) of each cell group was analyzed by a
3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltertrazolium bromide (MTT)
reduction method (FIG. 12). As a result, it was confirmed that,
when the anti-EPB41L5 monoclonal antibody was injected, the cell
viability decreased in a concentration-dependent manner, suggesting
that the anti-EPB41L5 monoclonal antibody of the present disclosure
exhibits therapeutic or preventive effects against advanced cancer
or gastric cancer (FIG. 12).
CONCLUSION
[0200] Cell adhesion proteins play a critical role in cancer
metastasis. Accordingly, the possibility of developing therapeutic
agents against cancer metastasis using antibodies against cell
adhesion proteins has been discussed. The present inventors have
made efforts to develop a new therapy based on a new adhesion
molecule or complex related to metastasis, thereby completing the
present disclosure. In EMT (Epithelial to Mesenchymal Transition),
epithelial cells lose cell-cell junctions and cell polarity, and
the actin cytoskeleton is reorganized to enable the mesenchymal
phenotype. EPB41L5 is reported to interact with p120-catenin, which
destabilizes E-cadherin, and paxillin, a focal adhesion kinase. In
addition, it is known that EPB41L5 binds to MPPS, a Crumbs complex
component that negatively regulates cell polarity. Another junction
protein, .beta.-catenin, colocalizes with EPB41L5 in the
basolateral membrane of kidney epithelial cells, but its binding
has not been confirmed. FAK has an FERM domain in its N-terminus
and binds with ASAP1/AMAP1 and paxillin, another binding partner of
EPB41L5 in the C-terminus.
[0201] In the present disclosure, it has been found that EPB41L5 is
responsible for the poor prognosis of gastric cancer patients. In
this regard, it was demonstrated that EPB41L5 is highly expressed
in cancer cells. Meanwhile, since TGF.beta. is known to promote
gastric cancer metastasis, it was predicted that expression of
TGF.beta. would have relevance to lymph node metastasis and the
prognosis of cancer. As a result of analyzing the relevance between
TGF.beta. and EPB41L5, a large number of Smad-binding motifs were
identified on the EPB41L5 gene promoter.
[0202] Accordingly, it was confirmed that knockdown of EPB41L5
abrogates the TGF.beta.1-induced increases in mesenchymal
transition and GC cell migration, indicating that EPB41L5 is a
major factor in TGF.beta. signaling. Taking these results together,
it can be seen that knockdown of EPB41L5 can control the migration
and invasion of gastric cancer cells caused by
epithelial-mesenchymal transition through the
TGF.beta./Smad3/EPB41L5 pathway.
[0203] In the present disclosure, it was confirmed that EPB41L5 is
located on the cell surface and promotes the in vitro and in vivo
metastasis of gastric cancer cells, suggesting that an antibody
that binds specifically to EPB41L5 may be used as a therapeutic
monoclonal antibody. Accordingly, the EPB41L5 monoclonal antibody
was developed. It was confirmed that the EPB41L5 monoclonal
antibody effectively blocked the TGF-.beta.1-induced migration and
invasion of gastric cancer cells, and that increased lung
metastasis of EPB41L5-overexprressing cells was significantly
inhibited by the anti-EPB41L5 monoclonal antibody.
[0204] In addition, it was confirmed that the anti-EPB41L5
monoclonal antibody of the present disclosure significantly
inhibited EPB41L5 protein expression in each of gastric cancer,
lung cancer and breast cancer cell lines (in particular, gastric
cancer cell line) and decreased the viability of cancer cells.
[0205] Therefore, it can be seen that the EPB41L5 monoclonal
antibody of the present disclosure is a very effective method for
prevention, treatment, or inhibition of metastasis, of cancer,
particularly gastric cancer.
[0206] Although the present disclosure has been described in detail
with reference to the specific features, it will be apparent to
those skilled in the art that this description is only of a
preferred embodiment thereof, and does not limit the scope of the
present disclosure. Thus, the substantial scope of the present
disclosure will be defined by the appended claims and equivalents
thereto.
INDUSTRIAL APPLICABILITY
[0207] The features and advantages of the present disclosure are
summarized as follows:
[0208] (1) The present disclosure provides a monoclonal antibody
and a fragment thereof that recognizes EPB41L5 as an antigen and
binds specifically thereto.
[0209] (2) The present disclosure may be used to identify an
antibody that binds specifically to an epitope of EPB41L5, and an
antibody identified through this method functions to inhibit
EPB41L5 signaling and, and this function enables the antibody to be
effectively used as a vaccine or therapeutic agent against cancer
in which EPB41L5 is involved, particularly gastric cancer.
Sequence CWU 1
1
131733PRTArtificial SequenceEPB41L5 protein 1Met Leu Ser Phe Phe
Arg Arg Thr Leu Gly Arg Arg Ser Met Arg Lys1 5 10 15His Ala Glu Lys
Glu Arg Leu Arg Glu Ala Gln Arg Ala Ala Thr His 20 25 30Ile Pro Ala
Ala Gly Asp Ser Lys Ser Ile Ile Thr Cys Arg Val Ser 35 40 45Leu Leu
Asp Gly Thr Asp Val Ser Val Asp Leu Pro Lys Lys Ala Lys 50 55 60Gly
Gln Glu Leu Phe Asp Gln Ile Met Tyr His Leu Asp Leu Ile Glu65 70 75
80Ser Asp Tyr Phe Gly Leu Arg Phe Met Asp Ser Ala Gln Val Ala His
85 90 95Trp Leu Asp Gly Thr Lys Ser Ile Lys Lys Gln Val Lys Ile Gly
Ser 100 105 110Pro Tyr Cys Leu His Leu Arg Val Lys Phe Tyr Ser Ser
Glu Pro Asn 115 120 125Asn Leu Arg Glu Glu Leu Thr Arg Tyr Leu Phe
Val Leu Gln Leu Lys 130 135 140Gln Asp Ile Leu Ser Gly Lys Leu Asp
Cys Pro Phe Asp Thr Ala Val145 150 155 160Gln Leu Ala Ala Tyr Asn
Leu Gln Ala Glu Leu Gly Asp Tyr Asp Leu 165 170 175Ala Glu His Ser
Pro Glu Leu Val Ser Glu Phe Arg Phe Val Pro Ile 180 185 190Gln Thr
Glu Glu Met Glu Leu Ala Ile Phe Glu Lys Trp Lys Glu Tyr 195 200
205Arg Gly Gln Thr Pro Ala Gln Ala Glu Thr Asn Tyr Leu Asn Lys Ala
210 215 220Lys Trp Leu Glu Met Tyr Gly Val Asp Met His Val Val Lys
Ala Arg225 230 235 240Asp Gly Asn Asp Tyr Ser Leu Gly Leu Thr Pro
Thr Gly Val Leu Val 245 250 255Phe Glu Gly Asp Thr Lys Ile Gly Leu
Phe Phe Trp Pro Lys Ile Thr 260 265 270Arg Leu Asp Phe Lys Lys Asn
Lys Leu Thr Leu Val Val Val Glu Asp 275 280 285Asp Asp Gln Gly Lys
Glu Gln Glu His Thr Phe Val Phe Arg Leu Asp 290 295 300His Pro Lys
Ala Cys Lys His Leu Trp Lys Cys Ala Val Glu His His305 310 315
320Ala Phe Phe Arg Leu Arg Gly Pro Val Gln Lys Ser Ser His Arg Ser
325 330 335Gly Phe Ile Arg Leu Gly Ser Arg Phe Arg Tyr Ser Gly Lys
Thr Glu 340 345 350Tyr Gln Thr Thr Lys Thr Asn Lys Ala Arg Arg Ser
Thr Ser Phe Glu 355 360 365Arg Arg Pro Ser Lys Arg Tyr Ser Arg Arg
Thr Leu Gln Met Lys Ala 370 375 380Cys Ala Thr Lys Pro Glu Glu Leu
Ser Val His Asn Asn Val Ser Thr385 390 395 400Gln Ser Asn Gly Ser
Gln Gln Ala Trp Gly Met Arg Ser Ala Leu Pro 405 410 415Val Ser Pro
Ser Ile Ser Ser Ala Pro Val Pro Val Glu Ile Glu Asn 420 425 430Leu
Pro Gln Ser Pro Gly Thr Asp Gln His Asp Arg Lys Cys Ile Pro 435 440
445Leu Asn Ile Asp Leu Leu Asn Ser Pro Asp Leu Leu Glu Ala Thr Ile
450 455 460Gly Asp Val Ile Gly Ala Ser Asp Thr Met Glu Thr Ser Gln
Ala Leu465 470 475 480Asn Asp Val Asn Val Ala Thr Arg Leu Pro Gly
Leu Gly Glu Pro Glu 485 490 495Val Glu Tyr Glu Thr Leu Lys Asp Thr
Ser Glu Lys Leu Lys Gln Leu 500 505 510Glu Met Glu Asn Ser Pro Leu
Leu Ser Pro Arg Ser Asn Ile Asp Val 515 520 525Asn Ile Asn Ser Gln
Glu Glu Val Val Lys Leu Thr Glu Lys Cys Leu 530 535 540Asn Asn Val
Ile Glu Ser Pro Gly Leu Asn Val Met Arg Val Pro Pro545 550 555
560Asp Phe Lys Ser Asn Ile Leu Lys Ala Gln Val Glu Ala Val His Lys
565 570 575Val Thr Lys Glu Asp Ser Leu Leu Ser His Lys Asn Ala Asn
Val Gln 580 585 590Asp Ala Ala Thr Asn Ser Ala Val Leu Asn Glu Asn
Asn Val Pro Leu 595 600 605Pro Lys Glu Ser Leu Glu Thr Leu Met Leu
Ile Thr Pro Ala Asp Ser 610 615 620Gly Ser Val Leu Lys Glu Ala Thr
Asp Glu Leu Asp Ala Leu Leu Ala625 630 635 640Ser Leu Thr Glu Asn
Leu Ile Asp His Thr Val Ala Pro Gln Val Ser 645 650 655Ser Thr Ser
Met Ile Thr Pro Arg Trp Ile Val Pro Gln Ser Gly Ala 660 665 670Met
Ser Asn Gly Leu Ala Gly Cys Glu Met Leu Leu Thr Gly Lys Glu 675 680
685Gly His Gly Asn Lys Asp Gly Ile Ser Leu Ile Ser Pro Pro Ala Pro
690 695 700Phe Leu Val Asp Ala Val Thr Ser Ser Gly Pro Ile Leu Ala
Glu Glu705 710 715 720Ala Val Leu Lys Gln Lys Cys Leu Leu Thr Thr
Glu Leu 725 73026PRTArtificial SequenceEPB41L5 epitope 2Ile Thr Pro
Ala Asp Ser1 53270PRTArtificial SequenceHuman EPB41L5 antigen 386
to 637th amino acid residue 3Glu Arg Arg Pro Ser Lys Arg Tyr Ser
Arg Arg Thr Leu Gln Met Lys1 5 10 15Ala Cys Ala Thr Lys Pro Glu Glu
Leu Ser Val His Asn Asn Val Ser 20 25 30Thr Gln Ser Asn Gly Ser Gln
Gln Ala Trp Gly Met Arg Ser Ala Leu 35 40 45Pro Val Ser Pro Ser Ile
Ser Ser Ala Pro Val Pro Val Glu Ile Glu 50 55 60Asn Leu Pro Gln Ser
Pro Gly Thr Asp Gln His Asp Arg Lys Cys Ile65 70 75 80Pro Leu Asn
Ile Asp Leu Leu Asn Ser Pro Asp Leu Leu Glu Ala Thr 85 90 95Ile Gly
Asp Val Ile Gly Ala Ser Asp Thr Met Glu Thr Ser Gln Ala 100 105
110Leu Asn Asp Val Asn Val Ala Thr Arg Leu Pro Gly Leu Gly Glu Pro
115 120 125Glu Val Glu Tyr Glu Thr Leu Lys Asp Thr Ser Glu Lys Leu
Lys Gln 130 135 140Leu Glu Met Glu Asn Ser Pro Leu Leu Ser Pro Arg
Ser Asn Ile Asp145 150 155 160Val Asn Ile Asn Ser Gln Glu Glu Val
Val Lys Leu Thr Glu Lys Cys 165 170 175Leu Asn Asn Val Ile Glu Ser
Pro Gly Leu Asn Val Met Arg Val Pro 180 185 190Pro Asp Phe Lys Ser
Asn Ile Leu Lys Ala Gln Val Glu Ala Val His 195 200 205Lys Val Thr
Lys Glu Asp Ser Leu Leu Ser His Lys Asn Ala Asn Val 210 215 220Gln
Asp Ala Ala Thr Asn Ser Ala Val Leu Asn Glu Asn Asn Val Pro225 230
235 240Leu Pro Lys Glu Ser Leu Glu Thr Leu Met Leu Ile Thr Pro Ala
Asp 245 250 255Ser Gly Ser Val Leu Lys Glu Ala Thr Asp Glu Leu Asp
Ala 260 265 270421RNAArtificial SequenceEPB41L5 siRNA (EPB41L5
siRNA#1) 4gcaauuggca gcuuauaauu u 21523RNAArtificial
SequenceEPB41L5 siRNA (EPB41L5 siRNA#2) 5uucagauucg ugccuauuca guu
2368PRTArtificial SequenceHeavy chain CDR1 6Gly Tyr Thr Phe Thr Ser
Tyr Trp1 577PRTArtificial SequenceHeavy chain CDR2 7Ile Tyr Pro Gly
Asn Ser Asp1 5812PRTArtificial SequenceHeavy chain CDR3 8Thr Arg
Gly Gly Lys Leu Pro Phe Ala Met Asp Tyr1 5 10911PRTArtificial
SequenceLight chain CDR1 9Gln Ser Leu Val His Ser Asn Gly Asn Thr
Tyr1 5 10103PRTArtificial SequenceLight chain CDR2 10Lys Val
Ser1119PRTArtificial SequenceLight chain CDR3 11Ser Gln Ser Thr His
Val Pro Trp Thr1 512119PRTArtificial SequenceHeavy chain_variable
region 12Gln Val Gln Leu Lys Glu Ser Gly Thr Val Leu Ala Arg Pro
Gly Ala1 5 10 15Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Thr Ser Tyr 20 25 30Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly
Leu Glu Trp Ile 35 40 45Gly Ala Ile Tyr Pro Gly Asn Ser Asp Thr Ser
Tyr Asn Gln Lys Phe 50 55 60Lys Asp Lys Ala Lys Leu Thr Ala Val Thr
Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Thr Asp
Glu Ala Ser Ala Val Tyr Tyr Cys 85 90 95Thr Arg Gly Gly Lys Leu Pro
Phe Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Ser Val Thr Val
Ser Ser 11513112PRTArtificial SequenceLight chain_variable region
13Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly1
5 10 15Asp Gln Ala Ser Met Ser Cys Arg Ser Ser Gln Ser Leu Val His
Ser 20 25 30Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly
Gln Ser 35 40 45Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser
Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Leu Gly Val
Tyr Phe Cys Ser Gln Ser 85 90 95Thr His Val Pro Trp Thr Phe Gly Gly
Gly Thr Lys Leu Glu Ile Lys 100 105 110
* * * * *
References